Three fourth-generation antigen/antibody combination assays (Elecsys, AxSYM, Architect HIV) and two third-generation (AxSYM, Centaur) HIV antibody immunoassays were evaluated. The evaluation panel of 479 samples included: nine tissue culture derived HIV-1 strains at four different p24 antigen concentrations (n=36), a p24 antigen sensitivity panel (n=10), 149 HIV-1 or HIV-2 confirmed antibody positive samples, ten anti-HIV-1 positive low titer samples, three seroconversion panels (n=21), and 253 HIV negative samples. The Architect had the best sensitivity for detection of HIV-1 antigen across eight HIV-1 subtypes, followed by the AxSYM while the Elecsys could not detect the highest antigen concentration evaluated (25 pg/mL) for eight of nine virus isolates. All assays showed 100% sensitivity for detection of HIV-1, group M, group O, and HIV-2 antibody positive samples. The Architect Ag/Ab Combo assay detected the first positive bleed of the three seroconversion panels and detected infection 4-26 days earlier than the third generation assays. Based on evaluation of 253 negative samples, assay specificity varied from 98.0% to 99.6%. The Architect HIV Ag/Ab Combo exhibited the best performance for specificity and detection of p24 antigen leading to closure of seroconversion window and demonstrating its utility for early diagnosis of HIV infection.