The integrity of the paternal genome is of paramount importance in the initiation and maintenance of a pregnancy in vivo and in vitro. The presence in the embryonic genome of chemical modifications at the level of DNA nucleotides and/or DNA strand breaks coming from the paternal genome (that have not been repaired by the oocyte after fertilization), is not compatible with normal embryo and fetal development. DNA fragmentation in human spermatozoa has been recently shown to be an important contributing factor to the etiology of unexplained infertility. In this review, the mechanisms responsible for DNA fragmentation in sperm, including defects in chromatin remodeling during the process of spermiogenesis, apoptosis and ROS-induced damage during sperm migration from the seminiferous tubules to the epididymis, are discussed. Also, the different methodologies used to determine DNA fragmentation in human sperm (with special emphasis on the SCSA test) and the applications of the SCSA test in the diagnosis and treatment of infertility, are presented. Finally, answers to common questions asked about the SCSA test are also included.