Many gene regulatory processes depend on proteins that interact with DNA. Characterizing these interactions can shed light on the molecular mechanisms that allow cells to control which RNA is made, when it is made, and how much is made. Additionally, DNA protein interactions are essential for cell division, DNA replication, and repair. Chromatin ImmunoPrecipitation followed by sequencing (ChIP-seq) has been an invaluable tool for understanding gene regulatory processes in the eukaryotic kinetoplastid parasite Trypanosoma brucei by mapping genomic binding sites for a protein of interest. We recently sought to expand the repertoire of available techniques to interrogate protein-DNA interactions by optimizing Cleavage Under Targets and Release Using Nuclease (CUT&RUN) for use in Trypanosoma brucei. The protocol presented here details a CUT&RUN protocol suitable for proteins in small complexes for which there is an available antibody.