Three-dimensional DNA fluorescent in situ hybridization (3D-FISH) has been extensively used for the study of chromosome organization, from large-scale chromosome territories to finer sub-megabase structures, such as topologically associating domains (TADs) or internal TAD features such as chromatin loops. The development of oligo-based technology has rendered this technique highly versatile and allowed to study chromosomal structures at different scale levels, as well as structural variations such as deletions, duplications, or translocationsTranslocation, as those occurring in cancerCancer and chromothripsisChromothripsis. Here, we present a comprehensive Oligopaint FISH protocol, from probe design and production to FISH, microscopy, and specific analysis that can be used with various cell lines.