Methotrexate is a chemotherapeutic and immunosuppressive drug used in the treatment of cancer, psoriasis, rheumatoid arthritis and several other disorders. Its use predisposes to hepatotoxicity which is a serious side effect. The current study aimed to assess the oxidative-stress-causing potential of methotrexate to liver cells and evaluate the hepatoprotective activity of the potent antioxidant astaxanthin, by affecting the oxidative stress biomarkers GSH (glutathione), SOD (superoxide dismutase), CAT (catalase) and MDA (malondialdehyde). A model of methotrexate-induced liver toxicity was employed on male rats. Thirty-six rats were divided into six groups; a negative control group, methotrexate induction group given (20 mg/kg) I.P. on day 13, three groups pretreated with oral astaxanthin in ascending doses (50, 75 and 100 mg/kg) for 14 days before methotrexate, and a conventional therapy group pretreated with silymarin (200mg/kg). The use of methotrexate significantly decreased liver tissue GSH, SOD and CAT while significantly increasing MDA, compared to the negative control. On the other side, astaxanthin used in all three doses significantly normalized these biomarkers. This study revealed that since astaxanthin significantly increased GSH, SOD and CAT while decreasing MDA that minimizes oxidative stress, it could be used as pretreatment in patients treated with methotrexate to decrease its hepatotoxicity.