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Rifampicin (RIF) is broadly used in the world for the treatment of tuberculosis, but the hepatotoxicity is still a major concern during clinical therapy. Studies showed that RIF induced oxidative stress activity in liver and directly toxic by it-s metabolites. Ocimum americanum (OA) contains phenols, flavonoids, and tannis which act as antioxidants and provides protection against free radicals caused by oxidative damage to cellular components that regulate the formation of ROS molecules. This study attempts to determine the hepatoprotective activity of ethanol extract of Ocimum americanum L. against RIF–induced liver damage in mice. Thereafter, the level serum of biochemical parameters, alanine transaminase (ALT) and aspartate transaminase (AST) were investigated. Treatment groups (RIF 300mg/kgBW + OA 2,8 mg/20grBW and RIF 300mg/kgBW + OA 5,6 mg/20grBW) were compared to RIF group (RIF 300 mg/kgBW) and normal control group. All treatments were given intragastrically for 14 days. Treatment with RIF significantly increased the activities of ALT and AST levels. OA-treatment reduced these activities in both extract dosage groups. However, a significant decrease was only found in serum ALT level. These findings indicate that the ethanol extract of Ocimum americanum L. exerted significant hepatoprotector effects, likely related to its antioxidant compounds.