Objective: The rate determination of cellular proliferation and cell viability is critical to the assessment of the effects of drugs on cells and there are different standard methods in evaluation of cell proliferation. Calprotectin is a cytosol metaloprotein in the neutrophil, macrophase, lymphocyte and monocyte cytosol that induce growth-inhibitory against various cell types including tumor cells. These finding indicate it can be as a therapeutic agtent in cancer diseases. Biological characteristics of calprotectin are contained antibacterial, antifungal, immonoregulation, chemotactic activity and inhibition of cell proliferation. In the present study, two staining assays; trypan blue and MTT in vitro evaluation of human calprotectin proliferation inhibition on human gastric cancer cells was compared. Materials and Methods: Calprotectin was purified from human neutrophil by chromatography methods. The human gastric adenocarcinoma cell line (AGS) was used. These cells were incubated in RPMI 1640 medium supplemented with 10% FBS in a humidified incubator (37oC & 5% CO2). AGS cells (10000 cell per well) were incubated to different concentration of calprotectin (25, 50, 100, 200, 400 mg/ml) at 24, 48, 72 h. In this study trypan blue dye exclusion and MTT assay were used for evaluation of cell proliferation inhibition.Results: Results of cytotoxicity effect of calprotectin on AGS cells show that there are significant correlation between two methods (P<0.01) and LC50 value of calprotectin calculated by MTT assay is lower than trypan blue in all time intervals. The LC50 of calprotectin for AGS cell at 24, 48 and 72 h was determined 33.29, 71 and 141.8 mg/ml by trypan blue and 96.78, 38.66 and 9.86 mg/ml by MTT assay, respectively. There are significant correlation between both methods at different time intervals and it is positive linear (P<0.01–P<0.001). Conclusion: Result of cell cyto toxicity of calprotectin by both methods indicated that correlation is positive and significant. It can be said that MTT assay is more sensitive, easy to handle, a large number of probes can be assayed in a relatively short time.