The determination of monoclonal antibody specificity is dependent upon the availability of purified antigen. Such material is not always available and this has proven to be one of the rate-limiting steps in monoclonal antibody production. The aim of the present study was to develop a generic approach to defining antibody specificity that bypassed the need for pure antigens through the use of proteomics. The scheme and its application to several biological mixtures are described. The results demonstrate the ability of the approach to identify antibodies against both the major components and the minor contaminants of a protein mixture. This approach should markedly enhance the characterization of antibodies to complex antigen mixtures.