The present study was designed to evaluate the effect of addition of different concentrations of ascorbic acid, glutathione and melatonin on motility and viability of frozen-thawed buffalo spermatozoa. In this experiment, frozen-thawed buffalo spermatozoa were in vitro cultured or 3 h in Fret-TALP medium (control), or Fret-TALP supplemented with 25, 50 and 100 µM ascorbic acid; 2, 3- or 5-mM glutathione; or 10-4, 10-5 and 10-6M melatonin and sperm motility and viability were determined every hour. Results showed that addition of 10-5 M melatonin to Fret-TALP medium can support sperm motility and viability up to 3 h of incubation. In conclusion, addition of melatonin to IVF medium of buffalo spermatozoa improves sperm motility and viability