The sperm chromatin compaction state refers to the tightly packaged structure in which the sperm DNA is specifically organized, unlike other cell types. The high grade of compaction in the sperm nucleus is crucial for preserving the integrity of the paternal genome during the journey from the testicle to the fertilization sites in many mammal species. The chromatin compaction results from complex transformations during spermiogenesis, in which protamines replace histones increasing DNA compaction. Chromomycin A3 (CMA3) staining is an indirect method to evaluate the sperm chromatin structure. An excess of histones (protamination defects) or a lack of disulfide cross-link causes an increase in the CMA3 fluorescence. Evaluated by flow cytometry, CMA3 staining enables quick detection of defects in the chromatin structure compaction. In this chapter, we described the method and some adaptations to perform this technique in mammal spermatozoa.