Epstein-Barr virus (EBV), a human herpesvirus, affects up to 95% of adults. Diagnosis of acute EBV infection can be challenging and often relies on the serologic antibody pattern to 3 distinct antigens, most often determined by indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assays (ELISAs), and, more recently, multiplex assays. We compared a multiplex assay for the simultaneous detection of antibodies to viral capsid (VCA), nuclear (EBNA), and early (EA) EBV antigens with ELISAs using IFA for discrepancy resolution. Concordance of the multiplex assay was good for all 4 antigens: VCA IgM, 86.6% vs ELISA and 92.9% vs IFA; VCA IgG, 92.8% vs ELISA and 98.0% vs IFA; EBNA IgG, 90.3% vs ELISA and 98.1% vs IFA; and EA IgG, 83.8% vs ELISA and 92.8% vs IFA. After IFA resolution, correlation between the multiplex assay and ELISA for serologic disease stage, based on the antibody profile of all 4 analytes, was 90%. The multiplex assay showed good correlation with an established ELISA and even better correlation with the "gold standard" IFA. Advantages of the multiplex assay over traditional methods include multiple results per assay, inclusion of internal controls for each assay, and well-to-well monitoring of assay drift.