The single color IFN-gamma ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-gamma secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-gamma resulted in a decreased magnitude of IFN-gamma but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-gamma and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.