HIV-1 RNA has become the standard for monitoring antiretroviral therapies. Dogma predicts, however, that a viral protein like p24 should be at least as good a marker of HIV disease activity, provided that it is measured with sufficient sensitivity and accuracy. Simple modifications including use of a more efficient virus lysis buffer, heat-mediated destruction of antibodies interfering with antigen detection, and tyramide signal amplification for increased sensitivity have highly improved the HIV-1 p24 antigen assay. The p24 antigen assay is inferior to RT-PCR in detecting viral particles, but the presence of extraviral p24 antigen in most samples makes largely up for this. p24 antigen testing is similarly sensitive and specific in diagnosing pediatric HIV infection, in predicting CD4+ T cell decline and clinical progression at early and late stage of infection, and suitable for antiretroviral treatment monitoring in both adults and children. Notably, p24 antigen was measurable even in patients with stably suppressed viremia, and its concentrations were correlated negatively with the concentrations of CD4+ T cells and positively with the concentrations of activated CD8+ T cell subsets. p24 antigen is an excellent marker of HIV expression and disease activity and can be used in the same fields of application as HIV RNA is used. The test is validated for subtype B, but requires further studies for non-B subtypes.