A greater understanding of the interaction between Streptococcus pyogenes, cells of the adaptive immune system, and the capacity to develop lasting immunity is needed to create an effective vaccine. Recent evidence has highlighted the potential importance of cell-mediated immunity against some S. pyogenes antigens. Whole-blood stimulation enables analysis of both innate and adaptive immune responses by identifying antigens that induce S. pyogenes-specific T cell responses. Human whole blood and PBMCs were stimulated with a range of S. pyogenes antigens, and Polymyxin E was added to mitigate residual endotoxin. Cytokine production was measured by intracellular staining, and in assay supernatants, by multiplex panels and ELISAs. Results were compared to circulating IgG against homologous antigens. Cellular activation was assessed by flow cytometry. Inflammatory cytokines were elicited in response to several antigens with varying magnitude. Superantigens, M protein, and SpyCEP produced some of the highest responses. Interferon-γ, an important link between innate and adaptive immunity, was produced in response to fewer antigens, although SpyCEP elicited high responses in healthy adults. Flow cytometric analysis of T cell activation and cytokine secretion suggested a pronounced memory CD4+ T cell response, supporting the potential for these assays to measure protective immunity. Understanding cellular and cytokine pathways involved in systemic immune responses to S. pyogenes is essential to bridge knowledge gaps in host-pathogen interactions and advance vaccine development. An in-tube assay measuring cytokine release in response to S. pyogenes antigen stimulation will simplify and streamline assessment of cell-mediated immunity to S. pyogenes, ideal for vaccine trials.