Abstract In a vaccine trial, assays for vaccine immunogenicity if performed locally will strengthen local site, can save costs and avoid hurdles associated with specimen transport. Here we report the optimization and validation of an Intracellular Cytokine Staining (ICS) assay which was undertaken in preparation for a phase I HIV vaccine trial conducted in Thailand. Intra-, and inter-operator variability were easily established. However, while attempting to set population cut offs for a positive response we found 4/36 (11%) high background responses of IFNγ + and/or IL-2 + CD8 + T cells (>1%) in normal healthy volunteers. The determinates of these unexpected responses were explored and minimized.