Many cellular signaling and communication events take place at the plasma membrane and thus the characterization of the plasma membrane proteome has been a hot research area in the hopes of learning more about these processes. Membrane microdomains are large protein and lipid complexes found on the cell surface membrane, able to concentrate or recruit signaling molecules or factors. The first step of any organelle proteomics study is to get a pure and enriched protein sample yet this has always been problematic in membrane proteomics as it is virtually impossible to purify a specific membrane type to homogeneity. In this review, we summarize the biochemical and proteomic approaches that have been used recently in the isolation and identification of several membrane microdomains and non-typical membrane proteins.