Post-transcriptional control is well established as a key mechanism of gene regulation in trypanosomatids. However, recent studies suggest that transcriptional regulation may also play a role, challenging long-standing dogma. The Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) provides a genome-wide overview of chromatin accessibility (i.e., whether chromatin is more open or closed), without requiring prior knowledge of chromatin markers which may be absent or poorly understood in trypanosomatids. Here, we present an optimized ATAC-seq protocol for use in Trypanosoma brucei, which has been used in both bloodstream and procyclic forms, and can be used to inform application of the method to other Euglenozoa. We also provide guidance on bioinformatic analysis, including integration of output files with established differential accessibility and RNA-seq data analysis pipelines.