HIV testing is the essential entry point for both treatment and prevention. The need to identify acute HIV infection (the period immediately after HIV acquisition, when persons are most infectious) and HIV-2 infection, which does not respond to many first-line antiretroviral agents, poses challenges for the traditional algorithm of Western blot confirmation after a repeatedly reactive antibody screening test. Immunoassays that detect antibodies earlier, tests for HIV RNA, and combination assays that screen simultaneously for both p24 antigen and HIV antibody are now approved for HIV diagnosis by the Food and Drug Administration. A revised testing algorithm can address the challenges posed by acute infection, HIV-2 infection, and the shortcomings of the Western blot. These new diagnostic strategies will allow earlier more accurate identification of infected persons so that they can benefit from effective treatment and also enhance abilities to focus prevention efforts where HIV transmission is most active.