BACKGROUND Human African trypanosomiasis (HAT) occurs as a result of infection with the protozoan parasites Trypanosoma brucei gambiense and T.b. rhodesiense and is nearly always fatal without treatment. However, current therapeutic options are severely limited and there is a desperate need for new compounds to treat the disease. Whole-cell high-throughput screening (HTS) is a technique frequently used to identify compounds with trypanocidal activity. METHODS The authors examine the development of whole-organism HTS assays for T.b. brucei. The authors describe the successes achieved through HTS and discuss the advantages and disadvantages of whole-organism HTS. CONCLUSIONS Despite hundreds of trypanocidal molecules being identified by whole-organism HTS, very few have progressed into preclinical development. The failure of molecules identified by HTS to progress along the drug development pathway is due to a multitude of factors including undrug-like molecules and molecules having poor pharmacodynamics/kinetic properties. Future studies should focus on screening libraries that contain drug-like molecules that possess some of the properties required in the final compound.