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Background: Liver is not only involved in maintaining homeostasis but also exhibits significant role in metabolism and detoxification of various drugs and toxins. Aim: To explore the hepato-protective role of N-acetylcysteine against methotrexate induced hepato-toxicity. Study design: Randomized controlled trial. Methodology: This study having mice (n=18) was carried out after ethical review committee’s (ERC) approval at Foundation university medical college in collaboration of National institute of health, Islamabad in 2017. Single intraperitoneal injection (20mg/kg) of methotrexate induced hepato-toxicity. Hepatoprotective effect was assessed by oral administration N-acetylcysteine (50mg/kg) alone with methotrexate. The extent of liver damage and effect of protective agents were determined by measuring serum ALT, AST, ALP after 24 hours of respective treatment. Liver samples were taken for histological analysis. One way ANOVA followed by Post Hoc Tukey test was applied for multiple comparisons of biochemical markers between the groups. Histopathological findings were analyzed by Chi Square test. p < 0.05 was considered significant. Results: Significant (p < 0.05) hepatotoxicity was seen with substantial elevation in serum ALT, AST and ALP with methotrexate. N-acetylcysteine attenuated the methotrexate induced hepatotoxicity significantly (p < 0.05). The histopathological examination showed mild steatosis along with focal pleomorphism in the liver of mice that received methotrexate in comparison to group treated with N-acetylcysteine and methotrexate though minimal inflammation was seen. Conclusion: We concluded that N-acetylcysteine ameliorates the methotrexate induced hepatotoxicity on when used concomitantly. Keywords: Hepatotoxicity, N-acetylcysteine and Methotrexate