Detergent removal from mixed micelles was combined with preparative size exclusion chromatography (SEC) on Sephacryl S 500 HR to prepare unilamellar and spherical liposomes of defined sizes between 50 and 100 nm with a very narrow size distribution (RSD of vesicle diameter between 13% and 25%). For neutral phosphatidylcholine and negatively charged phosphatidylcholine/phosphatidylglycerol liposome preparations, efficient sizing at the preparative scale was demonstrated by analyzing isolated SEC peak fractions with cryo-transmission electron microscopy and dynamic light scattering. The number-weighted average vesicle diameters obtained using both methods are in very good agreement for fractions of low polydispersity.