Chagas disease is one of the most lethal diseases in Latin America, but currently it has become a global health problem because of migration. It is caused by the parasite Trypanosoma cruzi (T. cruzi), an intracellular flagellated protist whose life cycle has been defined in detail, although the immunopathology of its infection is still quite unknown. Moreover, there are differences in pathogenicity between the strains in mice, Y produces a high lethality while VFRA remains in the host in a most chronic manner. Comparative proteomics studies between T. cruzi strains have not been performed extensively, they should allow the detection of relevant virulent factors or distinctive functions. We focused on the proteome analysis of trypomastigotes of Y and VFRA strains by mass spectrometry followed by gene ontology analysis to display similarities or differences in cellular components, biological processes and molecular functions. Also, we performed metabolic pathways enrichment analysis to see the most relevant pathways in each strain. There were a 20% of different proteins showing that Y strain has specific proteins for replication and growth, while VFRA for localization or transport, among others. We found a very similar profile in the different ontology groups, but with some punctual differences. We detected enriched antioxidant defenses in VFRA that could correlate with its behavior of chronic infection in mice controlling the ROS production, while Y strain displayed a great enrichment of pathways related with nucleotides and protein production, explaining the high parasite load and lethality of this strain.