qPCR functional screening rapidly detects the reactivity of melanoma antigen specific T cells in short term sensitized peripheral blood cultures. PBMC from 17 HLA-A2+ melanoma patients were plated in replicate microwells (n = 24) containing ~300,000 cells and individually sensitized for 6 days with either 1 μM of (A.) FLUM1, (B.) MART27–35, (C.) gp100209–217, (D.) gp100154–162, or (E.) no peptide (DMSO) in the presence of IL-2 (90 IU/ml). On day 6, a sample from every microculture (~100,000 cells) was screened using the qPCR assay for T cell recognition of the respective sensitizing peptide versus the HIVpol peptide pulsed onto T2 cells. Stimulation Index (SI) = IFN-γ mRNA (peptide x)/IFN-γ mRNA (HIVpol). (O) represents the SI for each microwell. Bar is median SI value. (*), not done. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0).