A 26‐color staining panel was developed to profile human antigen‐specific T cells in an intracellular cytokine staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal‐associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross‐validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.