The integral genome of sperm is crucial for successful fertilization and embryonic development. Different techniques have been used to determine the integrity of DNA in which acridine orange (AO) assay is easy and less costly. Though used in many species but the variability exists in technique. Therefore, the present study was aimed to optimize the steps of AO staining protocol for buffalo sperm which has not been explored yet. Semen was collected from two mature buffalo bulls, diluted in tris citrate egg yolk based extender and cryopreserved using standard protocol. The semen straws were thawed at 37oC and stained using different protocols as followed: Protocol 1 (washing effect); washing either with normal saline (NS) or phosphate buffered saline (PBS), Protocol II (heating effect during incubation in tampon solution); smears were immersed in tampon either at room temperature or at 60oC, Protocol III (concentration of dye in staining solution); slides were stained with 1, 5, 200, 500, or 1000µg/mL solution. The slides were observed, intensity of staining and debris presence in samples. The debris presence was reduced (P<0.05) due to washing of semen sample. Intensity of staining increased (P<0.05) due to washing and heating during incubation in tampon solution. However, single and double washing and type of buffer did not affect the staining intensity. Higher concentration (1000µg/mL) stained the fragmented DNA better than lower (P<0.05) concentrations. However, double stranded DNA was stained with lower concentration of dye in staining solution. In conclusion, the buffalo sperm should be stained with higher concentration of AO stain in washed sample either with NS or PBS and heated incubation in tampon solution for better assessment of fragmented DNA.