We developed an intracellular cytokine staining assay to quantify dengue specific memory T cells elicited by a primary dengue virus (DEN) infection. Peripheral blood mononuclear cells (PBMC) of volunteers who received experimental live attenuated monovalent DEN vaccines were stimulated with glutaraldehyde-inactivated dengue-infected Vero cell culture lysates from all four DEN serotypes. CD4+ T cell frequencies to previously identified MHC class II peptides were equivalent to 40-70% of the responses using virus infected cell lysates in two donors. IFN-gamma responses to DEN were detected from 0.04% to 0.45% CD4+ T cells. The highest IFN-gamma response was elicited by antigens from the homologous serotype. We detected serotype-specific and -cross reactive CD4+ T cell cytokine responses from all donors. This assay is suitable for measuring DEN specific CD4+ T cells in human PBMC from large population studies where donor haplotype is unknown or highly variant.