Aim:Evaluation of the effect of acetaminophen (APAP) overdose on biochemical parameters in buffalo calves.Materials and Methods:The experiment was conducted on six healthy male buffalo calves of 6-12 months age. The APAP was administered intramuscularly at the dose rate of 250 mg/kg body weight (B.W.) on day 0, followed by two subsequent doses at the dose rate of 50 mg/kg B.W. on day 2 and 4, respectively. Biochemical parameters including alanine aminotransferase (ALT), alkaline phosphatase (ALP), amylase, blood urea nitrogen (BUN), creatinine, and total acid phosphate were estimated in the plasma samples collected on 0, 1, 2, 3, 4, 5, and 6th day from the start of treatment.Results:Significant increase in the plasma levels of ALT (446.0%), ALP (137%), BUN (216.8%) and creatinine (149.2%) was recorded on day 3, 4, 3, and 4, respectively, after the start of APAP dosing. However, a significant decrease was observed in amylase activity with a maximum decline of 48.3% on 6th day after the start of treatment. No significant alteration was observed in ACP activity after APAP overdose.Conclusion:Administration of APAP in overdose produced hepatic dysfunction as evidenced by a significant increase in the activities of ALT and ALP, whereas reduced amylase may indicate acute pancreatitis in buffalo calves. In addition, repeated dosing also resulted in renal impairment in these animals as seen by a significant elevation in BUN and creatinine levels, whereas negligible effect on prostatic function.