Since the purpose of the spermatozoa is to contribute to the embryo with the haploid component of the male, the chromatin structure is a critical element for the success of fertilization and embryo/conceptus development and, most importantly, regarding the offspring viability. The assessment of the sperm chromatin by flow cytometry after DNA denaturation and acridine orange staining is the pioneering technique being validated in many species. Invented by D. Evenson and registered under Sperm Chromatin Structure Assessment (SCSA®), the technique has been applied in many contexts and is considered the golden standard for evaluating sperm DNA fragmentation. Whereas often forgotten, the technique provides relevant information about chromatin compaction or maturity. These parameters have been related to sperm fertility in humans and some non-human species, although there is controversy about their predictive value. This chapter revisits this technique and its protocol and describes some adaptations that could improve its performance under specific circumstances.