Giant unilamellar vesicles (GUVs) are a desired membrane‐mimetic system for the study of many membrane‐related phenomena, the function of MPs and the creation of synthetic cells. These micrometer sized vesicles are similar in size to bacteria and eukaryotic cells, and thus mimic these organisms more closely in terms of surface and volume. The size allows the integration of complex systems and entire metabolic processessuch astranscription and translation, as well asthe investigation of individual vesicles using light microscopy techniques, potentially cutting the costs of purified MPs needed to perform experiments by a factor of 100 compared to bulk methods. This makes them a very attractive system to investigate MPs, for example to test and develop novel drugs, and to create a bottom‐up synthetic cell. However, lipids do not spontaneously assemble into cell‐sized vesicles which has prompted the development ofseveral different techniquesfor GUV formation. For the same reason, these vesicles are more fragile towards the use of detergents, which complicates MP reconstitution. To harness the power of light microscopy measurements, GUVs have to be immobilized to enable real‐time observation over several minutes to hours. Lastly, the success of measuring MP function in a GUV also depends on the choice of detection system. In previous work in our lab, GUV electroformation on indium‐tin‐oxide (ITO) coated glass slides and reconstitution of MPs using charge‐mediated fusion of oppositely charged vesicles was established. GUVs were immobilized using a streptavidin‐biotin system to enable measurement of MP function. One of the disadvantages of GUV electroformation on ITO coated glass slides is the poor compatibility with high ionic solution, which could result in low protein activities due to formation at non‐physiological conditions. One of the aims of this project wasto establish GUV formation under physiologically relevant conditions to allow formation in buffer compositions optimal for MP function. We thus compared previously established electroformation on ITO coated glass slides with electroformation on platinum (Pt) wires and the more recently developed polymer assisted swelling using PVA. We observed that both Pt wire and PVA formation produced GUVs using various buffer compositions and that polymer assisted swelling produced a high yield of GUVs without much optimization, showing the potential and versatility of this method. Interestingly, we discovered that the immobilization was affected by the buffer composition, and that strong adhesion can lead to leakage and loss of encapsulated cargo, especially in PVA GUVs. This is an important finding as MPs are frequently followed using encapsulated fluorescent dyes, showing that immobilization conditions have to be tuned according to the buffer composition to provide sufficient immobilization while preventing too much cargo loss. Protons play an important role in many cellular processes, they are involved in many transmembrane transport reactions as well as in the production of ATP by the ATP synthase. Thus, GUVs should be able to maintain a proton gradient. Our measurements suggest that this is indeed the case also in immobilized vesicles that have not leaked vi encapsulated fluorophores. We further presented a simple strategy that could be used to estimate the protein concentration in GUVs after charge‐mediated reconstitution by fusion of GUVs with small vesicles containing labeled lipids and labeled MP. We could show that GUVs with more lipid‐coupled dye signal also showed more MP‐coupled dye signal, which could simplify the quantification of MPs in GUVs after fusion by following lipid‐coupled dye incorporation without the need for MP labeling. However, this strategy is not able to distinguish between simple adhesion or hemifusion of small vesicles and full fusion, which would be required for functional reconstitution of a MP. However, the same is true for simply following labeled MP signal, meaning that potentially other methods such as content mixing assays would be needed to get a better idea on the amount of functionally reconstituted MP. Finally, knowledge gained from the characterization of GUVs was applied for the reconstitution and measurement of cytochrome c oxidase from Rhodobacter sphaeroides using carboxyfluorescein and pyranine (HPTS). The former produced only weak and unclear signals and was prone to fast bleaching. Using ratiometric dyessuch as HPTS, pH calibration can be performed, where the observed ratiosshould be independent of dye concentration and bleaching. This proved to be challenging in GUVs, as the vesicles size seemed to have an effect on the observed HPTS ratio. Despite that, cytochrome c oxidase measurements using HPTS yielded better results and by characterization of vesicle shape and measurement of lipid‐coupled dye signal introduced via fusion, a correlation between the proton translocation and the relative amount of MP per vesicle could be observed, showing that thorough characterization of the GUVs can help to relate vesicle activities. Nonetheless, the different sizes of the vesicles were a considerable challenge for data analysis. We thus plan to use monodisperse GUVs produced by microfluidic techniques in collaboration with members of the deMello group form the ETH Zürich and we could show that these GUVs are fusogenic and can be potentially used to measure proton translocation. In a second project, we used bifunctional DNA duplexes to establish a new tool for the measurement of MP function. By linking pH‐sensitive dyes via DNA oligomers to cholesterol moieties, fluorescent probes can be anchored to the lipid membrane, allowing more efficient encapsulation compared to soluble dyes which could safe costs using precious probes. Addition of DNase I allows fast and simple removal of probes facing the liposome exterior, which are exposed to the buffered solution and thus do not contribute to the measurement of proton translocation in or out of the vesicle, a common problem with lipid‐coupled probes. Incorporation of bifunctional DNA into GUVs was slightly more challenging. Depending on the GUV formation method, as well as lipid and buffer composition, differences in the degree of incorporation into the membrane were observed, ranging from no membrane localization to complete incorporation. Formation furtherseemed to be negatively affected by the probe. Thus, further optimization of the probe might be needed to enable measurement of MP function in GUVs.