Cutaneous Leishmaniasis (CL) is an endemic disease in developing countries. Glucantime has been recommended for CLtreatment by world health organization, there are some restrictions in this case including high expense, side effects, frequent injections need, and incomplete efficacy. Considering different methods which have been used for disease treatment thus far; in present study, the zinc sulphate (ZS) effect on rural strain CL’ Viability of old world in vitro is under investigation. Iranian endemic species including Leishmania (L) major [MRHO/IR/75/ER] was appropriately provided, in different intervals, parasite numbers were counted in mentioned culture through two methods including the slide and cell proliferation ELISA. Finally, the considered counting was compared with the controlling group. Parasite species showed sensitivity to ZS. In vitro and in comparison with the controlling group, their numbers were reduced. ZS with 0.5%, 1%, 2%, and 3 % of density was added to the culture containing parasite and through the first to fifth days, the live parasite numbers in aforesaid culture was counted through the slide method by Neubar slide. The findings were analyzed statistically and level revealed P < 0.05. The findings revealed that all the cultures containing ZS (3%, 4%, 5%, and 6 %) in comparison with the controlling group showed a slower growth. The mentioned difference was statistically analyzed and revealed P < 0.05. Although ZS In vitro was affected, but probes are mandatory in the cases of animal and sick persons, figure out its daily dosage, concentration, time and duration.