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Proteoliposomes represent nanoscale assemblies of indispensable value for studying membrane proteins in general and membrane transporters in particular. Since no universal protocol exists, conditions for proteoliposome formation must be determined on a case-by-case basis. This process will be significantly expedited if the size and composition of the assemblies can be analyzed in a single step using only microliters of sample. Here we show that dual-color fluorescence cross-correlation spectroscopy (FCCS) is of great value for optimizing the reconstitution process, because it distinguishes micelles, liposomes and aggregates in heterogeneous mixtures and permits direct monitoring of the co-localization of proteins and lipids in the diffusing assemblies. As proof-of-principle, liposomes containing the functional multidrug resistance transporter NorA from Staphylococcus aureus were prepared, demonstrating that FCCS is an excellent tool to guide the development of reconstitution protocols.