High-throughput screening (HTS) of small-molecule libraries against pharmacological targets is a key strategy of contemporary drug discovery. This study reports a simple, robust, and cell-based luminescent method for assaying antimalarial drugs. Using transfection technology, we generated a stable Plasmodium falciparum line with high levels of firefly luciferase expression. A luciferase assay based on this parasite line was optimized in a 96-well plate format and used to compare with the standard [(3)H] hypoxanthine radioisotope method. The 50% inhibitory concentrations (IC(50)s) of chloroquine, artesunate, artemether, dihydroartemisinin and curcumin obtained by these two methods were not significantly different (P>0.05, ANOVA). In addition, this assay could be performed conveniently with a luminescence plate reader using unsynchronized stages within as early as 12h. Furthermore, the luciferase assay is robust with a Z' score of 0.77-0.92, which suggests the feasibility for further miniaturization and automation.