Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures.