Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) and outer monolayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 μm2 s−1 at 25 °C and D = 2.3 ± 0.7 μm2 s−1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.Read less <