Early detection of infection with human immunodeficiency virus (HIV) is critical for clinical diagnosis and treatment of patients, as well as for ensuring the safety of blood transfusion products. Recently, a number of fourth-generation HIV screening assays have been developed that offer increased sensitivity over earlier tests by combining detection of anti-HIV antibodies with detection of the p24 viral antigen. Previously, six different HIV assays were compared against a broad range of 30 seroconversion panels. In the present study, three of the newer fourth-generation assays were tested together with three of the third-generation HIV antibody-only assays. This extensive analysis highlights (i) the importance of p24 antigen detection for early diagnosis, (ii) the improved sensitivity of fourth-generation assays over antibody-only tests, and (iii) the superior performance of the Vidas Duo assay, which allows reduction of the diagnostic window by up to 2 weeks. Finally, the results emphasize the detection limitations of the different assays and suggest improvements for future HIV screening assays.