Male fertility can be affected by oxidative stress (OS), which occurs when an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to neutralize them arises. OS can damage cells and influence sperm production. High levels of lipid peroxidation have been linked to reduced sperm motility and decreased fertilization ability. This literature review discusses the most commonly used biomarkers to measure sperm damage caused by ROS, such as the high level of OS in seminal plasma as an indicator of imbalance in antioxidant activity. The investigated biomarkers include 8-hydroxy-2-deoxyguanosine acid (8-OHdG), a marker of DNA damage caused by ROS, and F2 isoprostanoids (8-isoprostanes) produced by lipid peroxidation. Furthermore, this review focuses on recent methodologies including the NGS polymorphisms and differentially expressed gene (DEG) analysis, as well as the epigenetic mechanisms linked to ROS during spermatogenesis along with new methodologies developed to evaluate OS biomarkers. Finally, this review addresses a valuable insight into the mechanisms of male infertility provided by these advances and how they have led to new treatment possibilities. Overall, the use of biomarkers to evaluate OS in male infertility has supplied innovative diagnostic and therapeutic approaches, enhancing our understanding of male infertility mechanisms.