ABSTRACTResistance to antimonial drugs has been documented inLeishmaniaisolates transmitted in South America, Europe, and Asia. The frequency and distribution of resistance to these and other antileishmanial drugs are unknown. Technical constraints have limited the assessment of drug susceptibility of clinical strains ofLeishmania. Susceptibility of experimentally selected lines and 130 clinical strains ofLeishmania panamensis,L. braziliensis, andL. guyanensisto meglumine antimoniate and miltefosine was determined on the basis of parasite burden and percentage of infected U-937 human macrophages. Reductions of infection at single predefined concentrations of meglumine antimoniate and miltefosine and 50% effective doses (ED50s) were measured and correlated. The effects of 34°C and 37°C incubation temperatures and different parasite-to-host cell ratios on drug susceptibility were evaluated at 5, 10, and 20 parasites/cell. Reduction of the intracellular burden ofLeishmaniaamastigotes in U-937 cells exposed to the predefined concentrations of meglumine antimoniate or miltefosine discriminated sensitive and experimentally derived resistantLeishmaniapopulations and was significantly correlated with ED50values of clinical strains (for meglumine antimoniate, ρ = −0.926 andP< 0.001; for miltefosine, ρ = −0.906 andP< 0.001). Incubation at 37°C significantly inhibited parasite growth compared to that at 34°C in the absence of antileishmanial drugs and resulted in a significantly lower ED50in the presence of drugs. Susceptibility assessment was not altered by the parasite-to-cell ratio over the range evaluated. In conclusion, measurement of the reduction of parasite burden at a single predetermined drug concentration under standardized conditions provides an efficient and reliable strategy for susceptibility evaluation and monitoring of clinical strains ofLeishmania.