The chemiluminescence method is the most commonly employed technique as a direct measurement of reactive oxygen species (ROS) generation by spermatozoa. This assay is capable of quantifying both intracellular and extracellular ROS. Moreover, the use of various probes allows for differentiation between superoxide and hydrogen peroxide production by spermatozoa. When the total antioxidant reserves are overwhelmed by excessive production of ROS, it results in oxidative stress. Therefore correct measurement of both ROS and total antioxidant capacity (TAC) is essential in the assessment of oxidative stress in sperm and semen. This chapter describes the methodological approach for measuring seminal oxidative stress through the use of chemiluminescence assay for accurate measurement of ROS and the colorimetric assay for measurement of TAC.