Identification of protein complexes is the key to understanding cellular functions. In this study, we present a novel method for the identification of multiprotein complexes from mammalian cells. By using the Strep-tag affinity chromatography method, enabling fast and simple one-step purification, coupled with competitive elution under physiological conditions, we successfully purified a PP2A holoenzyme protein complex from a cultured mammalian cancer cell line. We identified, by mass spectrometry, both known and novel interacting proteins for PP2A, and demonstrate that the purified PP2A complex is functional. The benefits and potential applications of the Strep-tag method for protein complex purification are discussed.