BACKGROUND: Plasmodium falciparum gametocytes, specifically mature stages, are the only stage in man transmissible to the mosquito vector responsible for malaria transmission. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria. The comprehensive profiling of in vitro activity of anti-malarial compounds against both early (I-III) and late (IV-V) stage P. falciparum gametocytes, along with the high throughput screening (HTS) outcomes from the MMV malaria box are described. METHOD: Two anti-gametocyte HTS assays based on confocal fluorescence microscopy, utilizing both a gametocyte specific protein (pfs16-GFP) and a viability marker (MitoTracker Red CM-H2XRos)(MTR), were used for the measurement of anti-gametocytocidal activity. This combination provided a direct observation of gametocyte number per assay well, whilst defining the viability of each gametocyte imaged. RESULTS: IC50 values were obtained for 36 current anti-malarial compounds for activities against asexual, early and late stage gametocytes. The MMV malaria box was screened and actives progressed for IC50 evaluation. Seven % of the "drug-like" and 21% of the "probe-like" compounds from the MMV malaria box demonstrated equivalent activity against both asexual and late stage gametocytes. CONCLUSIONS: The assays described were shown to selectively identify compounds with gametocytocidal activity and have been demonstrated suitable for HTS with the capability of screening in the order of 20,000 compounds per screening campaign, two to three times per seven-day week.