To advance T cell–based HIV vaccine development, it is necessary to evaluate the immune correlates of a protective CD8⁺ T-cell response. We have developed an assay that assesses the capacity ex vivo of HIV-specific CD8⁺ T cells to suppress HIV-1 infection of autologous CD4⁺ T cells. This assay directly reflects the ultimate effector function of CD8⁺ T cells, the elimination of infected cells, and accurately differentiates the effective CD8⁺ T-cell response in spontaneous HIV controllers from ineffective responses in other patients. In this article, we describe all the steps from cell purification to assessment of viral replication by HIV-p24 ELISA and analysis, along with conditions for cell culturing, and how to choose the viral infectious dose that gives the most reliable results. We also depict the conditions of a rapid assay on the basis of flow cytometry analysis of intracellular HIV-Gag products. These procedures take 14–17 d when the p24 ELISA assay is used, or 6 d with the intracellular Gag assay.