The assay has a lower quantitation limit, generally below 0.04%, depending on the cytokine subset. After optimization, the 8-color assay was validated for specificity, precision, linearity, limit of quantitation and robustness. We show that omission of viability dye staining results in an over-estimate of the true antigen-specific T cell response by up to two-fold. Here we describe extensive studies to optimize and quantitatively validate the 8-color ICS assay for use in clinical trials of candidate vaccines, which includes measurement of viable IFN-γ, IL-2, TNF-α and IL-4 secreting CD4+ and CD8+ T cells. Additionally, the use of banked cryopreserved PBMC samples makes this assay attractive in the setting of large efficacy trials where it is less feasible to perform immunoassays on freshly isolated samples. Intracellular cytokine staining (ICS) assays allow sensitive, quantitative ex vivo assessments of antigen-specific T cells including immunophenotyping of responding cells and measurement of multiple effector functions. Candidate HIV-1 vaccines currently being evaluated in clinical trials are designed to elicit HIV-1-specific cellular immunity.