The evaluation of the status of sperm chromatin is essential to deepen the male fertility study, but, despite its proven impact on fertility and the embryo, it is not routinely analyzed in andrology labs. One of the factors related to sperm functionality is the stability that disulfide bonds (DB) provide to the chromatin, which can be indirectly studied by analyzing the reduction of DB to free thiols. Monobromobimane (mBBr) is a fluorescent dye specific for the study of DB. It is a rapid and highly specific technique, easy to perform, and repeatable, and the total DB can be calculated from the direct fluorescent signal of free thiols. This chapter summarizes how to perform the mBBr technique in spermatozoa of multiple mammal species.