Leishmaniasis is a disease that covered under the title of neglected tropical diseases caused by protozoan parasites called Leishmania which can classify into three groups as visceral, cutaneous and mucocutaneous leishmaniasis. L. major is a type of parasite that causes cutaneous leishmaniasis and it is endemic in Iran, and Syria. However, cutaneous type leishmaniasis caused by L. major has been begun to detect in Turkey due to its close location to such countries. Moreover, the variety in the infectivity of L. major in a different region of Turkey has detected. Therefore, the uncertainty under the virulence effect of L. major is aimed to investigate. Large-scale protein analysis by mass spectrometry based proteomics has cleared up to proteome mapping for different organism recently. Generally, although two methodologies that involve gel-free and gel-based approaches have widely accepted for proteomic analysis, gel-free LC-MS/MS analysis were applied to characterize the proteome analysis of L. major parasite in two clinical case exhibiting passive and aggressive virulence effect on leishmaniasis. Finally, differential and common proteins that can affect the infectivity of L. major invastigated by shotgun analysis. As a result, samples showed that there are conflict results with the literature about GP63, secreted acid proteases, cysteine proteases and Peroxiredoxin proteins existence and also in the aggressive L. major cystathionine beta-synthase protein which has an role to synthesis of CPs and pyridoxal phosphate binding activity were proposed as a critical protein for L. major infectivity due to its association with SAPs, CPs.