An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-γ (IFNγ) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a ≤1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of ≥55 spots per 106 cells and ≥4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity.