Leishmaniosis is a tropical neglected parasitic disease that is endemic in many countries, including Middle East, with no existing effective vaccines. The bite of female sand-fly transmits the causative agent, Leishmania spp., to humans. High toxicity, resistance and treatment failure of the available chemotherapy against visceral leishmaniosis demands the investigation of new anti-leishmanial compounds. Lupeol is a form of triterpene isolated from several medicinal plants and possesses an antimicrobial property. In this study, cytotoxic effect of lupeol was screened against the mammalian amastigotes form and insect promastigote form of Leishmania donovani, following three cycles of incubation at different concentrations by MTT assay. Results revealed the in vitro anti-leishmanial effect of lupeol on both forms of the parasite where significant decline in promastigotes and amastigotes growth was observed. This was conducted along three times of follow up (24, 48, 72) hours, in comparison to the classical sodium stibogluconate treatment. Cell viability was calculated and the minimum IC50 was detected after 48 hours for amastigotes and 24 hours for promastigotes, 12.125 µM, 102.78 µM, respectively. Given the severity of visceral leishmaniosis and the toxicity of conventional chemotherapies, the anti-leishmanial activity of lupeol suggested a promising compound for additional clinical trials