Liposomes are commonly used as model membranes for studying the function of isolated membrane proteins. They are also used for reconstituting large membrane-associated protein complexes, such as the protein coats of intracellular transport vesicles. Here, we show examples of how dual-color fluorescence cross-correlation spectroscopy (or, more generally, fluorescence fluctuation spectroscopy) can serve to optimize in vitro reconstitutions and unravel protein-membrane interactions:(A) The reconstitution of detergent-solubilized, purified membrane proteins into liposomes is a prerequisite for a myriad of experimental studies, but experimental conditions generally have to be determined on a case-by-case basis. Fluorescence correlation spectroscopy (FCS) distinguishes micelles, liposomes and aggregates in homogeneous and heterogeneous samples based on their different mobilities. Dual-color fluorescence cross-correlation spectroscopy (dcFCCS), an extension of FCS, additionally detects the co-localization of protein and lipid in these diffusing entities, facilitating the optimization process [1]. The principle is not limited to proteoliposome formation, but can be applied more generally to reconstitutions into small diffusing membrane entities, such as liposomes or nanodiscs.(B) Studies of large, multi-subunit protein complexes, such as the COPII transport vesicle coat, require analysis of protein-lipid as well as protein-protein interactions. By virtue of their low perturbation, fluorescence techniques help optimizing complex reconstitution systems to obtain suitable preparations for cryo electron microscopy analysis of a complex structure [2], and help expanding the complexity of the reconstitution. The interaction of COPII components among each other and with model membranes are investigated.[1] Simeonov P, Werner S, Haupt C, Tanabe M, Bacia K, Membrane protein reconstitution into liposomes guided by dual-color fluorescence cross-correlation spectroscopy, Biophys. Chem. (2013), 184, 37-43.[2] Zanetti G, Prinz S, Daum S, Meister A, Schekman R, Bacia K, Briggs JAG, The structure of the COPII transport-vesicle coat assembled on membranes, eLife (2013), 2:e00951.