Early diagnosis of HIV infection and appropriate care reduces morbidity and mortality. As a result, recent guidelines recommend that HIV screening be routinely included in patient care. Routine screening will likely result in more patients being tested prior to seroconversion; fourth-generation assays can facilitate diagnosis in these patients. This study evaluated the performance of the automated fourth-generation ADVIA Centaur ® HIV Ag/Ab Combo assay. Samples from three sites were tested using the HIV Ag/Ab Combo assay and a CE-marked predicate assay. The HIV Ag/Ab Combo assay's relative sensitivity was 98.36% (599/609; 95% confidence interval: 97.00–99.21%), and the relative specificity was 99.74% (7743/7763; 95% confidence interval: 99.60–99.84%). The HIV Ag/Ab Combo assay detected seroconversion at the same bleed or at least one bleed earlier in 34/37 panels compared to the CE-marked predicate assay. Compared to the final result, the HIV Ag/Ab Combo assay's sensitivity was 100% (598/598; 95% confidence interval: 99.39–100.00%), and the specificity was 99.74% (7753/7773; 95% confidence interval: 99.60–99.84%). Sensitivity was 100% for the HIV genotypes tested. The HIV Ag/Ab Combo assay is a sensitive and specific assay that can assist clinicians in the early diagnosis of HIV infection.