The host immunologic response to group A streptococcal infections gives rise to numerous antibodies directed against cellular and extracellular bacterial antigens. For determining individual immune status, or studying the pathogenesis of group A streptococcal associated diseases, such as acute rheumatic fever (ARF), an assay capable of determining antibodies responses to multiple antigens would be of great advantage. We have developed a microsphere based, multiplexed immunoassay for the simultaneous quantitation of antibodies to nine different extracellular, ARF related tissue and group A streptococci specific antigens using only 5 microl of sample. Through the selection of microspheres and serum diluent, non-specific antibody binding was reduced by 17%. Different formulations of the coupling buffer were found to greatly influence the efficiency of coupling antigens to the carboxylated microspheres. Monoclonal antibodies against the different antigens demonstrated assay specificity as well as sensitivities of less than 1 ng/ml of antibody. This multiplexed assay should be a powerful research and clinical tool in determining antibody responses to group A streptococcal infections and in potentially determining the role of a variety of cross-reactive antigens in rheumatic fever and rheumatic heart disease.