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Metacyclic trypomastigotes are essential for the understanding of the biology of Trypanosoma cruzi, the agent of Chagas disease. However, obtaining these biological stages in axenic medium is difficult. Techniques based on charge and density of the parasite during different stages have been implemented, without showing a high efficiency in the purification of metacyclic trypomastigotes. So far, there is no protocol implemented where sepharose-DEAE is used as a resin. Therefore, herein we tested its ability to purify metacyclic trypomastigotes in Liver Infusion Triptose (LIT) medium cultures. A simple, easy-to-execute and effective protocol based on ion exchange chromatography on Sepharose-DEAE resin for the purification of T. cruzi trypomastigotes is described. T. cruzi strains from the Discrete Typing Units (DTUs) I and II were used. The strains were harvested in LIT medium at a concentration of 1 × 10⁷ …