Background Measles, mumps, rubella, and varicella-zoster (MMRV) immunity testing is important in occupational screening of healthcare workers and at-risk populations. Objectives The goal of this study was to compare the performance of the Bio-Rad BioPlex 2200 MMRV multiplex fluorescence immunoassay (MFI) against two enzyme immunoassay (EIA) methods: the Bio-Rad Evolis Twin Plus measles, mumps, and varicella-zoster (MMV) IgG assay and the Abbott Architect Rubella IgG assay. Study Design Clinically uncharacterized serum specimens were obtained and analyzed using the Bio-Rad BioPlex assay and compared against the EIA methods. Results The Bio-Rad BioPlex demonstrated total agreement of 85.5% (95% confidence interval (CI), 78.0 to 90.7%), 92.7% (95% confidence interval (CI), 86.7 to 96.1%), 92.4% (95% confidence interval (CI), 86.9 to 95.7%), and 98.8% (95% confidence interval (CI), 93.7 to 99.8%) for measles, mumps, varicella-zoster, and rubella, respectively, against the current EIA methods. Furthermore, precision testing agreed with the manufacturer's package insert in 10 of 13 pooled samples. Conclusion These data indicate that the Bio-Rad BioPlex has comparable performance to the EIA methods.