Malaria elimination/eradication campaigns emphasize interruption of parasite transmission as a priority strategy. Screening for new drugs and vaccines against gametocytes is therefore urgently needed. However, current methods for sexual stage drug assays, usually performed by counting or via fluorescent markers are either laborious or restricted to a certain stage. Here we describe the use of a transgenic parasite line for assaying drug sensitivity in all gametocyte stages.A transgenic parasite line expressing green fluorescence protein (GFP) under the control of the gametocyte-specific gene α-tubulin II promoter was generated. This parasite line expresses GFP in all gametocyte stages. Using this transgenic line, we developed a flow cytometry-based assay to determine drug sensitivity of all gametocyte stages, and tested the gametocytocidal activities of four antimalarial drugs.This assay proved to be suitable for determining drug sensitivity of all sexual stages and can be automated. A Z' factor of 0.79 ± 0.02 indicated that this assay could be further optimized for high-throughput screening. The daily sensitivity of gametocytes to three antimalarial drugs (chloroquine, dihydroartemisinin and pyronaridine) showed a drastic decrease from stage III on, whereas it remained relatively steady for primaquine.A drug assay was developed to use a single transgenic parasite line for determining drug susceptibility of all gametocyte stages. This assay may be further automated into a high-throughput platform for screening compound libraries against P. falciparum gametocytes.