OBJECTIVE To establish an assay using 9-color flow cytometry immunophenotyping to detect activation and apoptosis of human TCR Vβ lymphocyte subpopulations in peripheral blood samples. METHODS We used 5 antibodies (CD3, CD4, CD8, CD95, CD69), phospholipids binding proteins Annexin V, TCR Vβ Repertoire Kit and nucleus dye DAPI to establish a 9-color flow cytometry assay. Peripheral blood samples were taken from eight healthy people for test of antibodies and determination of optimal PMT and staining method (single-stained vs stained with all but one antibody). RESULTS Appropriate detecting voltage, antibody concentration and compensation methods were determined. The distribution of TCR Vβ subgroup in our samples was consistent with the TCR Vβ Repertoire Kit instruction and other published literature. CONCLUSIONS We have established a effective easy using 9-color flow cytometry immunophenotyping to detect human TCR Vβ lymphocyte subpopulations in peripheral blood samples.