Chronic liver disease and cirrhosis is the seventh leading causes of death in Taiwan in 2007. This indicates hepatoprotection is very important for Taiwanese. Nowadays, the model of carbon tetrachloride (CCl4)-induced hepatotoxicity is an official methodology used to evaluating the hepatoprotective effect of Health Foods in Taiwan. However, CCl4-induced hepatotoxicity is not common to people and CCl4 is recognized as a controlled-drug in Taiwan. Finding another chemical to replace CCl4 is necessary. Acetaminophen (APAP) is a clinically used analgesic and antipyretic drug, but overdose of APAP can cause liver injury in experimental animals and human. The first aim of this study was to establish the applicability of APAP-induced hepatotoxicity model in BALB/c mice for evaluating the hepatoprotective effect of Health Foods. Garlic (Allium sativum), a bulbous root with a strong flavor, is used widely in culinary preparations and as a folk medicine. It is rich in organosulfur compounds with biological activities, such as hepatoprotective, detoxifying and antioxidative activities. Previous studies showed that steam-distilled garlic oil (GO) gave hepatoprotective activity, but the dosage used was much higher than human’s consumption in daily life. Therefore, the second aim of this study was to evaluate the protective effects of lower dosage of GO on APAP-induced hepatotoxicity in BALB/c mice and to check the practicability of this model. The results indicated that the adequate doses of APAP to induce acute and chronic liver injury in BALB/c mice are 600 and 400 mg/kg bw, respectively. The administration of GO at the dose of 2.5 and 25 mg/kg bw could decrease ALT (alanine aminotransferase) and AST (aspartate aminotransferase) values in mice. GO also could modulate enzymes (glutathione S-transferase and cytochrome P450 2E1) of hepatic detoxification system, as well as increase hepatic glutathione contents and antioxidative enzymes (glutathione reductase, glutathione peroxidase and superoxide dismutase) activities. The hepatoprotective effect of GO was also confirmed in histopathological examination of the liver. In conclusion, the model of APAP-induced hepatotoxicity may be useful to evaluate the hepatoprotective effect of Health Foods. GO is a potential candidate as Health Food with liver protective function.