Abstract Background & aim: Easy access, rapid recovery and high potency of monocyte cell therapy have led to special attention in cell therapy research. Monocytes are considered as sticky cells to the flask. Therefore, finding the appropriate isolation method that has the least damage to the cell and its function is of particular importance. The purpose of this study was to compare the functional capabilities of monocytes after isolation with three methods: lidocaine / EDTA, trypsin and cold PBS / EDTA. Methods: In this experimental study, after extraction of peripheral blood mononuclear cells from Balb / c mice, cells (107 × 1 cells / ml) were incubated in RPMI culture medium in T25 culture flask for 4 hours. After incubation time, non-adherent cells (mainly lymphocytes) were separated by two rinsing and removed from the flask. Three different methods of trypsin, lidocaine and phosphate buffer saline were used for isolation of monocyte cells. After isolating the cells with each method, the functional capabilities of the monocytes were measured and compared with each other. The collected data were analyzed using one-way ANOVA. Results: The amount of extraction, survival, metabolic activity, phagocytosis percentage, respiratory explosion, nitric oxide levels, and yeast potential in cells isolated by lidocaine were significantly higher than other groups. Although the use of trypsin, although it results in the removal of more cells, is cooled to the PBS method, but these cells exhibit significant physiological impairment in comparison with the Lidocaine / EDTA or PBS / EDTA method. Neutral red uptake was detected by trypsin isolated monocyte cells in comparison to the other two methods at lower levels. In comparison between cold PBS and lidocaine, it seems that there is no significant difference between the monocytes obtained from the two methods in terms of neutralization. Conclusion: Compared to trypsin and PBS / EDTA, the Lidocaine / EDTA method is an appropriate method for isolating monocytes adherent to the flask, due to the extraction of more efficient and efficient cells. .