Accurately determining the number of peptide-MHC class I complexes on the cell surface is necessary when evaluating cellular processes or pharmaceuticals that alter the antigen presentation machinery. Here I describe a quantitative flow cytometry application for determining the number of peptide-MHC complexes on the surface of cells grown in tissue culture that express an endogenous protein from which the peptide is derived. The procedure requires a monoclonal antibody with the ability to distinguish MHC class I molecules presenting the peptide of interest from other peptide-MHC complexes. Fluorescence signal measured on antibody-labeled cells can be compared to fluorescent-calibrated beads to determine the relative number of antibodies bound to the cell surface and hence the number of specific peptide-MHC complexes expressed by the cell. As new monoclonal antibodies with TCR-like specificity for peptide-MHC complexes are created, this method will be helpful in quantifying the exact numbers of complexes generated by cell types and relating these numbers to physiological outcomes of T cell activation.