The presence of affinity reagents such as immunoglobulin in preparations for sensitive mass spectrometry analyses can preclude the identification of low-abundance proteins of interest. We report a method whereby antisera are purified and biotinylated prior to use in immunoprecipitation that allows for its efficient removal from proteomic samples via streptavidin capture. This method can similarly be extended to other affinity reagents such as recombinant fusion proteins for enhanced identification of interacting proteins.