Proteins typically act as components of poly-protein complexes, which may vary in composition and function, and may depend on subcellular localization and cellular state. To understand their biology, there is a need to identify the constituents in these various protein complexes. Conventional immunoprecipitation combined with proteomics analysis is nowadays routinely used to identify multiple interactors of a bait protein; however, it is unable to distinguish protein subcomplexes from one another. Blue native polyacrylamide gel electrophoresis is appropriate to separate individual protein complexes. Here, we describe a protocol that combines the specificity of immunoprecipitation, the size separating capacity of Blue native polyacrylamide gel electrophoresis, and subsequent mass spectrometry to delineate the protein constituents in subcomplexes of a targeted protein.