Trypanosoma cruzi, the protozoan that causes Chagas disease, possesses a complex life cycle involving different developmental stages. Experimental conditions for two‐dimensional electrophoresis (2‐DE) analysis of T. cruzi trypomastigote, amastigote and epimastigote proteomes were optimized. Comparative proteome analysis of the cell‐cycle stages were carried out, revealing that few proteins included in the 2‐DE maps displayed significant differential expression among the three developmental forms of the parasite. In order to identify landmark proteins, spots from the trypomastigote 2‐DE map were subjected to matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry peptide mass fingerprinting, resulting in 26 identifications that corresponded to 19 different proteins. Among the identified polypeptides, there were heat shock proteins (HSP; chaperones, HSP 60, HSP 70 and HSP 90), elongation factors, glycolytic pathway enzymes (enolase, pyruvate kinase and 2,3 bisphosphoglycerate mutase) and structural proteins (KMP 11, tubulin and paraflagellar rod components). The relative expression of the identified proteins in the 2‐DE maps of the T. cruzi developmental stages is also presented.