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Understanding antigen-specific T-cell responses is crucial for advancing immunotherapies and vaccine development. This study proposes a novel approach combining two complementary assays: the 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay (tracking proliferation over 0-48 h) and the VPD450 dye dilution assay (tracking proliferation over 4-6 days). Integrating these techniques provides additional insights into T-cell proliferation kinetics. Both assays were independently optimized using anti-CD3 and anti-CD28 polyclonal T cell stimulation. 1 μM VPD450 is suitable for assessing T-cell proliferation. The EdU concentration should match the stimulation strength, requiring higher concentrations to efficiently track DNA replication detection during increased cellular division. Day 5 was the optimal read-out day for the EdU incorporation assay. We then combined the VPD450 dye dilution and EdU incorporation assays. As a proof of principle, we stimulated PBMCs from healthy donors with tetanus toxoid to assess antigen-specific T-cell responses. Additionally, we demonstrated the assay's application in drug research by evaluating proliferation in a mixed lymphocyte reaction with abatacept, an agonistic anti-CTLA-4 antibody. This combined approach offers qualitative insights into T-cell proliferation kinetics, beneficial for assessing novel vaccine efficiency or for designing new treatments targeting T cell proliferation, such as in autoimmune settings., (© 2024 the Australian and New Zealand Society for Immunology, Inc.)