The chapter discusses the concept of reactive oxygen species (ROS) determination. Oxidative stress is one of the factors responsible for DNA fragmentation in sperm. Excessive generation of ROS in semen could be the cause of infertility. Low concentration of ROS affects mobility of the sperm and high concentration results in loss of viability of the sperm. The assessment of ROS may help in the treatment of male infertility by suitable antioxidant. This test determines the total ROS generated in human semen by leucocytes and spermatozoa.