Freezing and thawing processes have the potential to adversely affect post-thaw quality of semen. In the current study, we endeavored to ascertain the most favorable application of L-cysteine for cryopreservation of Achai and Holstein Friesian bull semen under the subtropical conditions of Peshawar region of Pakistan. Semen of six adult bulls, three Achai (the indigenous breed) and three Holstein Friesian (exotic breed) amount to the experimental material at Government Cattle Breeding and Dairy Farm Harichand, Charsadda. Separate artificial vagina was utilized for semen collection from either breed, maintained at 42°C temperature. Gross and microscopic examination was carried out subsequent to the semen collection. Semen specimen with more than 70% motile characteristics were used for further processing. The semen was split into four aliquots in which tris-citric acid extender (TCA) was supplemented with various concentrations of L-cysteine 0 mM, 0.5 mM, 1.0 mM and 1.5 mM. Cooling and equilibration of the diluted semen was done at 4°C for 4 h subsequently filled in straws at the same temperature and were cooled in liquid nitrogen vapors for 10 min. followed by storage at -196°C for assessment. Progressive motility and viability of spermatozoa were significantly higher in 1.5 mM and 1.0 mM L-cysteine supplemented groups in comparison with 0.5 mM or 0 mM. The number of supra-vital hypo-osmotic swelling test positive spermatozoa at 1.5 mM and 1.0 mM was greater in comparison with lower concentration of 0.5 or 0 mM L-cysteine. Furthermore, significant improvement for acrosomal integrity was recorded in semen samples treated with 1.5 mM L-cysteine followed by 1mM, 0.5mM. It is concluded that 1.5 mM is the most favorable concentration of L-cysteine to be added to the TCA extender for improving the post-thaw quality of semen of Achai and Holstein Friesian bulls under the existing environmental conditions.