Immunonaffinity chromatography is a powerful technique for rapid purification of proteins. In a single-step purification, it is possible to purify proteins for testing in model systems and for conducting enzyme kinetic studies. Because the immunoaffinity-purified proteins are typically >90–95% pure, depending on the starting material, interference from remaining contaminants is rare. This method describes an immunoaffinity chromatography technique for purifying proteins from over-expression in mammalian cell culture. The immobilization of the monoclonal antibody or polyclonal antiserum is presented. Conditions for purifying up to milligram quantities of protein are given, including a representative chromatogram.