Chromatin plays essential roles in all nuclear processes, and the dynamic regulation of its accessibility is critical for orchestrating gene expression programs. Chromosome conformation capture (Hi-C) combines chromosome conformation capture (3C) techniques with next-generation sequencing (NGS) to map physical interactions between distant chromatin regions, enabling genome-wide analysis of three-dimensional genome architecture. We previously conducted the first Hi-C analysis of Trypanosoma cruzi to explore its spatial genome organization and its potential role in gene expression regulation. This chapter describes the experimental procedures for preparing 3C and Hi-C libraries and computational tools for data analysis.