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The current Mycobacterium bovis BCG vaccine provides inconsistent protection against pulmonary infection with Mycobacterium tuberculosis (Mtb). In mice studies, immunity induced by subcutaneous BCG wanes by the time. The evidence for immunogenicity following BCG vaccination in Indonesia is limited. This preliminary study measured Mtb-specific T cell response in peripheral blood mononuclear cells (PBMC) using flow cytometry. Three designs were compared in the stimulation of PBMC with Mtb culture filtrate protein (CFP) and additional protein transport inhibitor Brefeldine A (BFA). They were 24 hours or 5 days of CFP stimulation with BFA at the last 4 or 18 hours. The intra-cytoplasmic cytokine staining (ICS) was then performed to assess the production of IFN-gamma, IL-2, and TNF by CD4+ T cells. Single production of IFN-gamma or TNF detected in the 24-hour was higher than that in the 5-day. The 18-hour BFA resulted in a significant proportion of polyfunctional CD4+ T cells. In conclusion, detection of Mtb-specific T cell response using ICS of human PBMC is optimum using 24 hours of antigen stimulation with additional BFA at the last 18 hours. The duration of antigen stimulation and protein transport inhibition affects the amount of intra-cytoplasmic cytokine response analysed using flow cytometry.