Objective: Although the interest in cryopreservation is increasing due to the increase of young cancer patients and infertility patients, there is a limit to cryopreservation. Cryopreservation itself can cause mechanical and chemical stress, ultimately leading to the formation of reactive oxygen species (ROS) and oxidative stress. ROS inhibits the expression of antioxidant enzymes produced in cells, resulting in increased DNA fragmentation and apoptosis. Identifying substances that reduce oxidative stress-induced damage to the ovarian tissue has garnered remarkable interest in recent years. Hence, we evaluated the degree of protection of the ovarian tissue against oxidative stress by cryopreservation with vitrification following treatment with N-acetylcysteine (NAC) and the Klotho protein. Methods: The control group and the cryopreservation groups were randomly assigned, and treated NAC, Klotho, or the combination (NAC + Klotho). The cell morphological change, DNA damage, senescence, and apoptosis of each group after the freeze-thaw process were compared. Results: Both NAC and Klotho were found to be more effective at protecting against DNA damage than the control; however, DNA damage was greater in the NAC + Klotho group than in the group treated with NAC and Klotho, respectively. DNA damage and cellular senescence were also reduced during the vitrification process when cells were treated with NAC, Klotho, or the combination (NAC + Klotho). NAC increased apoptosis during cryopreservation, whereas Klotho inhibited apoptosis and NAC-induced apoptosis. Conclusion: Therefore, Klotho may be an effective cytoprotective agent for the cryopreservation of ovarian tissue.