Whole-cell High-Throughput Screening (HTS) is a key tool for the discovery of much needed malaria transmission blocking drugs. Discrepancies in the reported outcomes from various HTS Plasmodium falciparum gametocytocidal assays hinder the direct comparison of data and ultimately the interpretation of the transmission blocking potential of hits. To dissect the underlying determinants of such discrepancies and assess the impact that assay-specific factors have on transmission-blocking predictivity, a 39-compound subset from the Medicines for Malaria Venture Malaria Box was tested in parallel against three distinct mature stage gametocytocidal assays, under strictly controlled parasitological, chemical, temporal and analytical conditions resembling the standard membrane feeding assay (SMFA). Apart from a few assay-specific outliers, which highlighted the value of utilizing multiple complementary approaches, good agreement was observed (average ΔpIC50 of 0.12 ± 0.01). Longer compound incubation times improved the ability of the least sensitive assay to detect actives by 2-fold. Finally, combining the number of actives identified by any single assay with those obtained at longer incubation times yielded greatly improved outcomes and agreement with SMFA. Screening compounds using extended incubation times and using multiple in vitro assay technologies are valid approaches for the efficient identification of biologically relevant malaria transmission blocking hits.