Methods that combine efficient solubilization with enrichment of proteins and intact protein complexes are of central interest in current membrane proteomics. We have developed methods based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3–10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offers removal of soluble proteins that can be further improved by manipulation of the driving forces governing protein distribution between the phases. Integral and peripheral membrane protein subunits from intact membrane protein complexes partition to the detergent phase while soluble proteins are found in the polymer phase. An optimized solubilization protocol is presented in combination with detergent/polymer two-phase partitioning as a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.