ABSTRACT Acetaminophen (APAP) is a widely-used analgesic, while toxic doses of which induce liver injury. Inducible cyclooxygenase-2 (COX-2) is derived prostaglandins which play an anti-inflammatory role in acetaminophen-induced hepatotoxicity. Selective activation of vascular endothelial growth factor (VEGFR1, flt -1) on endothelial cells increased mRNA levels of hepatocyte mitogens (IL-6) and hepatocyte growth factor leading to prosurvival effects on hepatocytes. The aim of this study was to compare the hepatoprotective effect of N-acetylcysteine (NAC; the antidote for APAP) with that of α-Lipoic acid (ALA) and/or Thymoquinone (THQ) either alone or in combination on liver injury induced by APAP. APAP administration elevated most of the previously measured parameters and decreased GSH, SOD, and total protein levels compared with the control group. Liver sections of H&E demonstrate liver injury characterized by centrilobular hepatocellular necrosis, COX-2, and flt-1 expressions were also increased. Treatment with all fore mentioned antioxidants ameliorated most of the altered parameters compared to APAP-treated group. Treatment with the combination of ALA and THQ was the most effective therapy in the attenuation of liver injury assessed by a decrease in ALT and ALP activities and down-regulation of COX-2 and flt-1 expression. Section of liver from rat received APAP, ALA and THQ shows a marked improvement of hepatic degeneration which restricted to few hepatocytes with mild vacuolation of their cytoplasm while the nuclei appear normal mimic to control cells. It was concluded that the natural antioxidants such as ALA and THQ, may be considered as a potential antidote in combating liver injury induced by APAP.