<p>Cryopreservation, an integral aspect of assisted reproductive technology, negatively affects the sperm motility, viability, membrane integrity, and DNA stability through the induction of cryo-oxidative stress and damage to cellular function. L-methionine, a sulfur-containing amino acid with significant antioxidant potential, was examined for dose-dependent effects on post-thaw sperm quality in 85 semen samples (n = 43) fertile and (n = 42) infertile. The Fertile and Infertile semen samples were cryopreserved in media supplemented with L-methionine (1.5 mM, M1; 3 mM, M2; and 6 mM, M3) in comparison with commercial cryopreservation medium (CM). After the thawing, sperm motility, morphology, viability, plasma membrane and acrosome integrity, oxidative stress (MDA), antioxidant enzyme activity including superoxide dismutase (SOD) and catalase, and DNA fragmentation were evaluated. The 3 mM L-methionine (M2) group significantly promoted spermatozoa motility, progressive motility, viability, morphology, and acrosome integrity(p &lt; 0.05) while reducing MDA levels and enhancing the activities of superoxide dismutase (SOD) and catalase (p &lt; 0.001) as a strong antioxidant protection. The DNA fragmentation was lowest in the M2 group in fertile and infertile men. In contrast, the highest concentration tested (6 mM, M3) did not improve sperm quality parameters and was associated with reduced motility, viability, and increased DNA fragmentation, suggesting potential toxicity at higher concentrations. The ROC analysis showed M2 (AUC: 0.88 vs. commercial medium) to be much more sensitive for the diagnosis of sperm damage. This study suggests that supplementation with 3 mM L-methionine may serve as a promising cryoprotective strategy for improving post-thaw sperm quality, particularly in infertile men.</p> Graphical Abstract <p></p>