Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes.