We report a method combining mixed lymphocyte reaction (MLR) using a carboxyfluorescein diacetate succinimidyl ester (CFSE)‐labeling technique, intracellular cytokine immunofluorescence staining (ICIS), and multiparameter flow cytometry for simultaneous determination of proliferation and cytokine‐secreting activity in T cells responding to allo‐stimulation. C57BL/6 (B6) mice and Balb/c mice were used in the experiments. CFSE‐labeled responder splenocytes were cultured with irradiated stimulator splenocytes, followed by ICIS. In both the Balb/c stimulator‐versus‐B6 responder (Balb/c‐vs.‐B6) and the B6‐vs.‐Balb/c allogeneic combinations, interleukin (IL)‐2 secreting cells and interferon (IFN)‐γ secreting cells were identified predominantly in proliferating CD4⁺ and CD8⁺ T cell fractions, respectively. The suitability of this method was proven by demonstrating a close relationship between the values of cytokines in culture supernatants (that were determined by Cytometric Bead Array assay) and indexes for cytokine‐production (that were obtained by multiplying the percentage of cytokine‐producing cells in T cells and mean fluorescence intensity of cytokine‐staining determined by the combined MLR and ICIS).