The magnitude and the quality of T-cell response are critical endpoints in the immune-monitoring of cancer patients. The release of cytokines and lytic factors by T cells operate in each phase of the cancer immunity cycle. The simultaneous cytokine production, defined as T-cell polyfunctionality, is a dynamic state of different T-cell subsets and may represent a surrogate biomarker of response to immune-mediated therapies. To quantify the T-cell immune mediators, different methodologies are available. Herein we describe two flow cytometry-based protocols to detect the simultaneous intracellular cytokine production, by using different methods of T-cell activation. Among the different procedures, multicolor flow cytometry is considered a powerful, quick, and semiquantitative technique, able to achieve the analysis of both surface and intracellular proteins, expressed by specific T-cell subsets. We report the capability of this technique to study simultaneous cytokine production within a heterogeneous population. As the field of novel single-cell high-throughput methodological approaches advance in this exciting era of Immuno-Oncology, we expect to gather even more information on the immunodynamics of polyfunctional T cells and their role in improving disease control.