Background: Sperm cryopreservation potential can be explored in diverse conditions to hamper future fertility. Reactive oxygen species (ROS) were among the prime candidate responsible for vulnerability of the sperms due to lack of intracellular antioxidants defense. Aim: The study aim to investigate the effect of cryopreservation on sperm biochemical profile and DNA fragmentation. Methods: This controlled observational hospital-based study was conducted on men participants (n=100). The semen specimens with normal sperm count and motility post fertility work up was collected in a sterile vial containing cryoprotectant and thereafter stored in a freezer having liquid nitrogen -196°C for 7 days at controlled rate. Sperm morphology, DNA fragmentation and motility along with malondialdehyde and total antioxidant capacity (TAC) were measured before and after cryopreservation. Results: Semen specimens post cryopreservation demonstrated significant decrease in sperm motility, number and malondialdehyde level (p<0.001). There was a significant increase in DNA fragmentation and total antioxidant capacity levels (p<0.001). Conclusion(s): Sperm cryopreservation may lead todeleterious variations of structure and function(s) due to generation of ROS that contribute in DNA fragmentation.