To maintain healthy nonhuman primates for use in biomedical research, animals are routinely screened for several infectious agents at most facilities. Commonly, monkey serum samples are tested by conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, for antibodies to specific infectious agents. For testing for antibodies against multiple agents in each sample, conventional immunoassays are laborious and time-consuming. More efficient immunoassays are needed. Accordingly, we have developed a novel multiplex serodiagnostic system based on individually identifiable, fluorescent microbead sets, where each bead set is coated with antigens from a purified preparation of a specific virus. The coated bead sets are mixed to enable the detection of antibodies to multiple viruses in one serum or plasma sample. These viruses include four agents that are routinely tested for maintenance of specific-pathogen-free monkeys, namely, simian immunodeficiency virus, simian type D retrovirus, simian T-cell lymphotropic virus, and herpes B virus, as well as simian foamy virus and rhesus cytomegalovirus, both of which are commonly found in nonhuman primates. This multiplex microbead immunoassay (MMIA) enabled the simultaneous detection of antibodies to all six viruses in single serum samples as small as 1 microliter. The results obtained by MMIA analysis correlated with results of conventional ELISAs, which detect antibodies to single agents. Thus, this multiplex microbead detection system is an efficient diagnostic modality for serosurveillance of nonhuman primates.