Non-internalized parasites were washed away, and compounds were added at various concentrations in a final volume of 100 µL. A dose-response curve for miltefosine was included in all assays as a quality control. Each condition was tested in quadruplicate. After 72 hours of incubation, the medium was replaced with 100 µL of PBS, and 25 µL of Glo-lysis buffer from the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA) was added.