Trypanosomatids express a diverse repertoire of small non-coding RNAs despite lacking the canonical RNA interference machinery in most species. These small RNAs—including fragments derived from tRNAs, rRNAs, and other non-coding sources—are thought to play regulatory roles in parasite biology and host–parasite interactions. In this chapter, we provide a detailed protocol for the isolation, library preparation, sequencing, and computational analysis of small RNAs in trypanosomes. The method is optimized to preserve short RNA species while minimizing artifacts such as adaptor dimers and concatamerization. It includes quality control and size selection steps to ensure the reproducibility and high quality of the libraries, allowing the study of the transcriptome of small RNAs in these parasites.