Introduction: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a safe, convenient, and low-cost technique with high applications for the measurement of cell proliferation rate in researches and clinical laboratories. Our aim was to evaluate the proliferation rate of human peripheral blood mononuclear cells (PBMCs) and production rate of Tumor necrosis factor alpha (TNF-α) by these cells after various mitogens stimulation in different situations. Methods: The MTT test was performed with various concentrations of mitogens including concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA) on the PBMCs. The cells were incubated for 24, 48, 72, and 96 hours in the culture medium and TNF-α cytokine assay was performed on the supernatant of the cultured splenocytes using the enzyme-linked immunosorbent assay (ELISA) method. Results: The optimal time and incubation of the PBMCs with the mixture of PHA-ConA were 5 μg/mL and 72 hours, respectively. The TNF-α level increased significantly after PHA-ConA and PHA stimulation. Conclusion:The results showed that the mixture of PHA-ConA (at the concentration of 5 μg/ mL) can give rise to the optimal results on stimulation of the PBMcs using the MTT assay after 72 hours incubation.