Cryopreseravation exerts damages to sperm including crystal formation, oxidative stress, osmotic shock and lipid peroxidation. α-Lipoic acid (ALA); an antioxidant that plays a role in ATP production and breakdown of free radicals thus reduces oxidative stress. The aim of this study was to optimize the concentration and effect of ALA on post-thaw quality and survival at 37°C of buffalo (Bubalusbubalis) bull sperm. Semen of 3 mature Nili Ravi breeding bulls was collected twice a week using artificial vagina. The ejaculates of 3 bulls were pooled bull wise (replicates = 6), divided into 6 aliquots, diluted with the Tris-citrate-egg yolk extender supplemented with different concentrations of ALA (0, 0.5, 1, 1.5, 2 and 2.5mM) and cryopreserved in 0.5-mL French straws using standard procedure. Two straws per replicate were thawed and pooled and incubated in modified synthetic fluid (mSOF) at 37°C in a humidified CO₂ incubator. Semen was evaluated for motility, plasma membrane integrity (PMI; hypo-ospmotic swelling test), viability and acrosomal integrity simultaneously (FITC-PNA/PI assay) and DNA integrity using acridine orange assay; at 0, 1.5, 3 and 4.5h of incubation. The data were presented as Mean ± SEM and analyzed 6 × 4 factorial repeated measure ANOVA for 6 concentrations and 4 times using MIXED procedure in SAS Enterprise Guide (version 4.2) taking time as repeated measure. The results revealed that extender supplemented 0.5 mM and 1 mM ALA resulted in higher (P < 0.05) sperm motility (55 ± 1.3% and 55.5 ± 1.3% respectively than control; 48.5 ± 1.37%, 1.5 mM; 49 ± 1.37% and 2 mM; 43.5 ± 1.37%), PMI (59.8 ± 1.8% and 60 ± 1.8% respectively), viability (67.3 ± 1.5% and 68.5 ± 1.5% respectively) and DNA integrity (99.29 ± 0.48% and 99.39 ± 0.48%) than control and other groups after thawing. The survival of sperm was also recorded higher (P < 0.05) due to 0.5 and 1 mM ALA resulting in motility (23.5 ± 1.3% and 25.5 ± 1.3%), PMI (34.3 ± 1.8% and 37.8 ± 1.8%), viability (38.6 ± 1.5% and 42.6 ± 1.5%) and DNA integrity (97.34 ± 0.48%and 98.0 ± 0.48%) than control and other groups at 4.5 h of incubation. In conclusion, α-lipoic acid enhances the post-thaw quality and survival of buffalo bull sperm.