The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects millions of people in Latin America. T. cruzi has a complex life cycle characterized by several developmental forms present in vertebrate and invertebrate hosts. In vertebrate mammalian hosts T. cruzi is found as intracellular amastigotes and bloodstream trypomastigotes. On the other hand, in the intestine of the insect (Reduvidae) vector epimastigotes and metacyclic trypomastigotes are the present forms. From all the developmental stages of the parasite, intracellular amastigotes are the most difficult to obtain in large amounts, since they are the only ones that grow inside the host cells. Here, we describe a simple and robust protocol that enables the isolation of either intracellular or extracellular amastigotes in large amounts. While intracellular amastigotes are obtained from infection of mammalian cell cultures, extracellular amastigotes are obtained by differentiating bloodstream trypomastigotes in axenic cultures. Both intracellular and extracellular amastigotes are purified by anion-exchange chromatography.