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Summary: Here, we describe a combined in cellulo and in vivo approach to identify compounds with higher potential for efficient inhibition of Trypanosoma cruzi. Phase I of in cellulo assays is designed to exclude inactive or toxic compounds, while phase II is designed for accurate IC50, CC50, and selective index (SI) determination. Compounds showing high SI are tested using in vivo infection models in parallel with benznidazole to assess their efficacy relative to a reference drug used for Chagas disease treatment.For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.