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Objective: Evaluation of free radical scavenging activity of Berberine (BBR) in acetaminophen (AAP) induced liver injury. Study design: Experimental study. Place and Duration: Animal House, Isra University Hyderabad from October 2015 to March 2016. Methodology: A sample of 80 male Wistar rats was selected according to inclusion and exclusion criteria and was divided into a control and three experimental groups. Acetaminophen, N-acetyl cysteine (NAC) and BBR were administered in standard doses. Cardiac puncture collected blood samples after 18 hours of the post-experiment period. Liver function test, antioxidant enzymes, and malondialdehyde (MDA) were detected by ELISA assay kit (Fortress Diagnostics). The data was analyzed on Statistix 10.0 software (USA) at 95% CI (P≤ 0.05). Results: The BBR showed antioxidant and antiperoxidant activity against acetaminophen-induced liver injury. BBR treated animals showed increased serum and tissue SOD, GPX, CAT, and GSSH with a reduction in tissue MDA (p=0.0001). Liver injury ameliorating effect of BBR was superior to N-acetyl cysteine. Conclusion: The present study suggests Berberine protects against acetaminophen-induced liver injury by its free radical scavenging activity.