Background: Cryopreservation of human semen alters the spermatozoal structure, resulting in a reduction in sperm function parameters. Various antioxidants may be able to slow or prevent this type of injury. Glutathione (GSH) has numerous antioxidant properties; supplementing the semen with GSH before freezing may assist in the restoration of post-thaw sperm functionality.Objective: To investigate the effect of adding 5mM of glutathione before freezing on human sperm cryosurvival.Materials and methods: Semen samples were collected from 30 patients (22 normozoospermic and 8 asthenozoospermic) after 3-5 days of sexual abstinence. Following liquefaction, macro- and microscopic examinations were performed. The samples were then divided into two equal aliquots: the first aliquot received 5 mM of glutathione before freezing, and the second aliquot was considered the control (without glutathione). The samples were frozen using the rapid cryopreservation method and then preserved for 7-10 days in a liquid nitrogen tank before being thawed. Thawed samples from each group were examined microscopically according to the WHO 2010 guidelines. Aniline blue was used to assess the maturity of the DNA. Then, the direct-swim-up technique was applied to thawed samples from both groups to select the best sperm quality.Results: The cryopreservation process negatively impacts all sperm characteristics in both groups. Sperm DNA integrity decreased. Glutathione addition before freezing decreased sperm chromatin immaturity (SCI) percent compared to the control (22.98 ± 0.83, 20.79 ± 0.56). The post-thaw performance of the swim-up technique resulted in a select sperm population with high progressive motility (p = 0.04) and an improvement in DNA integrity in the treated group versus the control group after thawing.Conclusion: The DNA integrity in normozoospermic samples was improved by adding 5 mM glutathione before freezing. Performing the swim-up technique after thawing had no effect on sperm quality in asthenozoospermic patients.