The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring. Damaged DNA in the single sperm that fertilizes the female egg can have a dramatic negative impact on fetal development. This comprehensive and detailed unit presents a rapid, reliable, practical test for DNA integrity based on staining with acridine orange. SCSA data have been conclusively shown to predict sub/infertility. This assay is ideally suited to human and animal fertility clinics to assess male sperm DNA integrity as related to fertility potential and embryo development. The authors, who have decades of experience in studying sperm viability, provide extensive commentary and methodological tips, making this unit the most detailed method for this test published to date.