Polyfunctional T cells that simultaneously produce the cytokines IFN-_γ, IL-2 and TNF have been correlated with better clinical outcomes in various diseases. To date, cytokine polyfunctionality within T cells has been exclusively studied by intracellular cytokine staining coupled with flow cytometric analysis. Thus, further downstream interrogation of polyfunctional T cell characteristics such as transcriptomic analysis has not been possible. Here, we report the use of a flow cytometric method based on cytokine secretion assay technology to detect and isolate, for the first time, viable human polyfunctional T cells directly from in vitro stimulated whole blood samples. We demonstrate the successful application of this method to sort polyfunctional T cells obtained from human volunteers, which can be then used for downstream applications such as transcriptomic analysis using RTqPCR. This assay will facilitate in-depth investigations of T cells with distinct cytokine polyfunctionality, including defining their molecular profile and understanding the mechanisms regulating their generation and function.