Egg yolk is inevitably associated with risks of microbial contamination and anti-cryoprotectant agents that necessitate the investigation of some synthetic alternatives.To investigate the potential of carboxylated poly-L-lysine (CPLL) as a replacement for egg yolk during the cryosurvivability of Nili-Ravi buffalo sperm.Semen collected from four Nili-Ravi buffalo bulls (two ejaculates / bull / day; total 40 ejaculates for five replicates) was cryopreserved in different experimental extenders viz: Control (CPLL 0%, egg yolk 20%); E1 (CPLL 5%, egg yolk 15%); E2 (CPLL 10%, egg yolk 10%); E3 (CPLL 15%, egg yolk 5%) and E4 (CPLL 20%, egg yolk 0%). Post-thaw quality was assessed in terms of sperm motility, plasma membrane integrity (PMI), viability, live:dead ratio, lipid peroxidation of sperm and total antioxidant capacity of seminal plasma.Sperm motility improved (P<0.05) in extenders replacing 5%, 10% and 15% egg yolk with CPLL. Sperm PMI, viability and live:dead ratio also improved (P<0.05) in extenders replacing 10%, 15% and whole (20%) egg yolk with CPLL. In contrast, sperm DNA integrity was not different (P>0.05) when CPLL replaced egg yolk at any level. The lipid peroxidation level decreased with a concomitant increase in total antioxidant activity of seminal plasma when CPLL replaced egg yolk at 5%, 10%, 15% and 20%.Replacement of 15% egg yolk in the extender with CPLL improves all sperm quality parameters: motility, PMI, viability, live:dead ratio, lipid peroxidation of sperm and total antioxidant activity of seminal plasma.