Oxidative stress, characterized by an imbalance between prooxidants and antioxidants in favor of the former, can lead to cellular damage due to the accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In spermatozoa, oxidative stress plays a crucial role in signaling pathways associated with capacitation, a process essential for fertilization. However, the limited capacity of spermatozoa to buffer ROS makes them susceptible to oxidative stress. Measurement of oxidative stress in spermatozoa is important as it precedes or directly causes the loss of basic sperm functions—such as motility and the membrane changes required for zona binding and gamete fusion—associated with fertility. This chapter aims to describe protocols for assessing oxidative damage in spermatozoa, encompassing the generation of ROS in mitochondria to the manifestation of oxidative damage in DNA, proteins, and lipids. In addition to the detailed instructions provided within this chapter, we have sought to provide background information on the development and significance of the assays which we most commonly utilize in our laboratory.