Enzyme-linked immunosorbent assay (ELISA) is normally used to quantify the amount of serum IgG antibodies against measles, mumps, rubella, and varicella-zoster virus (MMRV). However, this method is time- and material-consuming. Therefore, a multiplex immunoassay for the simultaneous quantitative detection of antibodies against MMRV was developed. In-house as well as commercially available antigens can be used, making the assay available for all laboratories. The multiplex assay is much more sensitive than the separate ELISAs and has a high specificity, and only 5 μl of serum is needed. Heterologous inhibition did not exceed 11.5%, while homologous inhibition varied between 91.3 and 97.9%. Good correlations with the in-house ELISAs for measles (R(2) = 0.98), mumps (R(2) = 0.97), and rubella (R(2) = 0.97) virus as well as with the ELISA kit for varicella-zoster virus (R(2) = 0.95) were obtained. In conclusion, the MMRV multiplex assay is a good alternative to the conventional ELISAs and suitable for use in serosurveillance and vaccine studies.