The present study aimed to establish a simple method to yield large amounts of Leishmania tropica amastigote-like forms in axenic cultures and to compare the superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymes at different stages of L. tropica. Different culture conditions were tested to find the optimum condition of axenic amastigotes generation. Superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were determined at logarithmic and stationary phases and axenic amastigote stage of the parasite. A high proportion (88%) of amastigote-like forms of L. tropica was observed in BHI medium supplemented with 20% FCS, pH 4.5, and incubated at 37ºC with 5% CO2. The results showed that SOD activity was at the lowest level in the logarithmic phase of promastigotes and increased towards the stationary phase of promastigotes and amastigote stage. The results showed that the optimum condition for differentiation of L. tropica promastigotes to axenic amastigotes was BHI medium containing 20% FCS at pH 4.5, incubated at 37ºC in the presence of 5% CO2. It seems that SOD, but not GPX is a major determinant of intracellular survival of the parasite.