Chagas´ disease is a neglected sickness endemic in Latin America, caused by the protozoa Trypanosoma cruzi. 1e current treatment for the disease is unsatisfactory, and the development of potent compounds for novel molecular targets is critical. In this framework, proteomics could be a powerful tool in the evaluation of possible candidates for drug intervention. In this work, a two-dimensional electrophoresis (2- DE) and mass spectrometry (MS) approaches were employed in T. cruzi epimastigotes (Y strain). Di8erent gel staining protocols (Coomassie Blue, Pro-Q-Diamond and Pro-Q-Emerald) were performed to assess the protein content and possible post-translational modi)cations of this parasite. Here, 78 most intense spots were identi)ed by Coomassie staining, 22 by Pro-Q-Diamond (phosphoproteins) and 15 by Pro-QEmerald (glycoproteins). Compared with the results of other large-scale T. cruzi proteomic studies, 15 novel proteins were identi)ed here using MALDI-TOF/TOF, and 12 of these have not yet been described at the protein level. Functional analysis of the identi)ed proteins pointed to protein metabolism, and the localisation prediction indicated cytosol as the most prevalent localisation of these proteins. Eight proteins presented no similarity to human sequences and thus represent a group of promising biomolecules for chemotherapy intervention. Our data provides novel insights in the metabolic pathways of T. cruzi, which could aid in the discovery of alternative drugs for Chagas´ disease.