Trypanosomatids are protozoan parasites, of interest due to both their disease burden and deeply divergent position within the eukaryotic lineage. The African trypanosome, Trypanosoma brucei, has emerged as a very amenable model system, with a considerable toolbox of methods available, including inducible overexpression, RNA interference, and a completed genome. Here we describe some of the special considerations that need to be addressed when studying trypanosome gene function, and in particular small GTPases; we provide protocols for transfection, RNA interference, overexpression and basic transport assays, in addition to an overview of available vectors, cell lines, and strategies.