We set out to develop a PSA peptide-loaded tetramer for enumeration of PSA-specific CD8(+) T cells in the Balb/c mouse model. A candidate major histocompatibility complex (MHC) class I PSA peptide (HPQKVTKFML(188-197)) was selected on the basis of its ability to restimulate PSA-specific CD8(+) T cells to secrete interferon-γ in our assays. Next, H-2L(d)-restricted peptide-loaded and fluorescently labeled tetramers were produced in conjunction with the NIH Tetramer Core Facility, Atlanta, GA, USA. This tetramer was then tested for staining specificity and optimized for detection of PSA-specific CD8(+) T cells induced by our PSA-encoding adenovirus tumor vaccine. The MHC class I PSA peptide demonstrated successful restimulation of CD8(+) T cells isolated from mice previously vaccinated with a PSA-encoding adenovirus tumor vaccine, with no restimulation observed in control-vaccinated mice. The peptide-loaded H-2L(d) tetramer exhibited the desired binding specificity and allowed for detection and frequency determination of PSA-specific CD8(+) T cells by flow cytometry. We have successfully designed and validated a PSA peptide tetramer for use in the Balb/c mouse model that can be used to test PSA-based prostate cancer vaccines. Until now, PSA-specific CD8(+) T cells in the mouse have only been detectable via cytotoxic T-lymphocyte assays or intracellular cytokine staining, which primarily assess antigen-specific functional activity and not their absolute number. This research tool provides laboratories the ability to directly quantitate CD8(+) T cells elicited by PSA-specific immunotherapies and cancer vaccines that are tested in mouse models.