In this study, different Trypanosoma cruzi assay protocols were evaluated in order to understand the key parameters influencing drug susceptibility. The assays compared in this study were an image-based intracellular T. cruzi assay quantified through an image-mining algorithm and an intracellular assay utilizing a β-galactosidase-expressing T. cruzi strain. This research was supported by the Drugs for Neglected Diseases initiative (DNDi)