Leishmaniasis is an infectious disease caused by Leishmania that widespread in 98 countries. The differentiation of Leishmania (L) from procyclic to metacyclic promastigote has occurred along with morphological and biochemical changes in proteome scale. We aim here to identify the proteomes of two successive developmental forms (procyclic and metacyclic promastigotes) from Leishmania major isolates using SWATH-MS quantitative proteomics technique. Isolated proteins from procyclic and metacyclic lysate were digested, fractionated and subjected to SWATH-MS. Proteins significantly different in abundance were analyzed using gene ontology (GO) and protein-protein interaction network (PPIN). Our study showed that 52 proteins were changed in abundance between the two consecutive developmental stages. Differentially expressed proteins were classified into nine classes by GO analysis. Significant modulations in translation, antioxidant and stress-related defenses, energy metabolism, structural and motility-related proteins were detected between procyclic and metacyclic stages. We found that elongation factor-2 and various structural constituents of ribosome were down-regulated during metacyclogenesis, while motility related proteins including ADP-ribosylation factor-3, paraflegellar rod protein-2C and tubulin alpha-chain were up regulated. According to network analysis, ENOL has been introduced as main hub-bottleneck protein and EF-1b, Hsp60 and GDH have been determined as seed proteins. Our results show that significant proteins in abundance are crucial features of metacyclogenesis in L. major. The protein function analysis illustrated that synthetic pathway involved proteins were down-regulated in metacyclic, which is the main feature of this stage of parasite growth cycle, while up-regulation of motility and energy metabolism related proteins is consistent with infective feature of metacyclic stage. Based on our results, we suppose that differentially expressed proteins possibly play a critical role in L. major differentiation. In addition, our finding demonstrated the possibility of SWATH-MS as viable technique to faster detect new stage-specific proteins in Leishmania and further studies are required for the validation of the results.