Extracellular vesicles (EVs) or exovesicles are a heterogeneous group of small cell-derived membranous structures that carry complex cargoes including lipids, proteins, RNA, and DNA. Emerging evidence suggest that EVs secreted by kinetoplastid parasites play a cardinal role in the pathogenesis of diseases they cause, becoming valuable structures for understanding parasite-host interactions. Moreover, the characterization of EVs molecular cargo may provide a new approach to develop alternative tools for diagnosis and therapy of infectious diseases. EVs have a potential use as biomarkers since it contains a repertoire of DNA species that could be detected at different stages of infection by PCR-based assays. Here, we provide a detailed protocol for the isolation of Trypanosoma cruzi-derived EVs and purification of its DNA cargo for subsequent characterization. The methods described here are transferrable to other medically important parasites that are well adapted to grow in vitro and, therefore, suitable volume of EVs-containing supernatants can be obtained.