Early and accurate diagnosis is essential for optimal therapeutic outcomes in patients infected with HIV. Currently, none of the commercially available fourth-generation assays differentiate HIV-1 and HIV-2 antibodies (Ab) or the HIV-1 p24 antigen (Ag). The aim of this study was to evaluate the performance of a novel assay, the BioPlex 2200 HIV Ag-Ab. This assay uses a multiplex flow immunoassay design allowing the simultaneous detection and identification of antibodies to HIV-1 (groups M and O), HIV-2, and the HIV-1 p24 antigen, in addition to providing a traditional composite result. A total of 1,505 routine serum samples were prospectively tested. Results were compared with those from the Architect HIV Combo assay. The sensitivity of the BioPlex 2200 was 100%. The specificity assessed on repeated false-positive samples was 99.5%. In addition, 524 frozen specimens from patients known to be infected with HIV-1 or HIV-2 were tested. Of these specimens, 420 were infected with HIV-1, including 156 of known genotypes, 86 were infected with HIV-2, 7 were infected with HIV-1 and HIV-2, and 11 were from patients with acute HIV infection. Sensitivity was 100% for the HIV genotypes tested. The differentiation capabilities of the BioPlex 2200 HIV Ag-Ab assay for HIV-1, HIV-2, dual HIV-1/HIV-2, and early infections were 100%, 90.7%, 100%, and 90.9%, respectively. The BioPlex 2200 is a sensitive and specific assay that offers advantages over conventional HIV combo assays, also referred to as fourth-generation assays, to accurately differentiate and report HIV-1 p24 antigen and HIV-1 and HIV-2 antibodies.