Our goal was to determine if a multiplex technique using a fluorescent bead-based flow cytometric assay could yield results comparable to traditional enzyme-linked immunosorbent assay (ELISA) in terms of sensitivity, specificity, cross-reactivity, and throughput. We applied both techniques to serologic screening of specific pathogen-free macaques, for type D simian retrovirus, simian T-lymphotropic virus, Cercopithicine herpesvirus 1, and simian immunodeficiency virus, and found a high correlation between the bead-based multiplex assay and ELISA. The multiplex assay demonstrated greater sensitivity with no loss in specificity when compared to the ELISA. A lower false-positive rate with the multiplex assay decreased the number of confirmatory Western blots required. Using the multiplex assay, we were able to screen samples for 4 viruses simultaneously in the time it took to perform a single-virus ELISA, resulting in a faster turnaround time and higher throughput. The multiplexed assay provided greater sensitivity, increased stability, and better performance than ELISA.