The protozoan parasites of the genus Leishmania are the causative agents of different clinical diseases. Fetal calf serum (FCS) is the main part and the most expensive ingredient of the Leishmania culture media. Here, the efficacies of different concentrations (1%, 2.5%, 5%, and 10%) of the filtered and autoclaved sheep and mouse urine were evaluated as a growth stimulator in Leishmania culture procedure. The results indicated that culture media enriched with the filtered sheep and mouse urine supported the growth of the parasites and can be used for cultivation of Leishmania parasites. In conclusion, this study has demonstrated an alternative low-cost medium that could be used in cultivation process of Leishmania major promastigotes.