Centella asiatica, is well known to be a valuable medicinal plant for producing valuable compounds such as asiaticoside, asiatic acid, madcasoside and madcasic acid. The plant is believed to improve memory, lower blood pressure, be a strong antioxidant and anticancer. Therefore, it is important to optimize tissue culture methods in order to facilitate the extraction of medicinal compounds, gene transfer as well as improvement of medicinal properties of the plant. Calli prepared from various medicinal plants can be used to increase the amount of medicinal compounds in the cell suspension culture and gene transfer. The aim of this study was to investigate the effect of different concentrations of two hormones, BAP and NAA, on leaf explant for callus initiation, as one of the important sources of secondary metabolites production. For this purpose, leaf explants were treated with 6 different concentrations of BAP (0, 0.5, 1, 1.5, 2.5, 3.5 mg/L) and 5 different concentrations of NAA (0, 0.25, 0.5, 1, 2 mg/L). The results of this study showed that the best callus was obtained by the combination 1.5 mg/L of BAP and 0.5 mg/L of NAA, resulting in the leaf explants with callus induction of 100%, fresh weight of 1.457 gr and callus diameter of 1.459 cm. The results showed that two hormones of BAP and NAA have synergistic effects on the increase of the quality and quantity of the produced calli.