Herbal medicines are gradually being accepted for their low toxicity and high efficacy, but their hepatotoxicity still needs to be recognized. For the multiple compounds in herbs, hepatocytes in vitro would be a useful tool for the evaluation of herbal hepatotoxicity. This study aimed at cocklebur/atractyloside-induced hepatotoxicity and the protective effects of glycyrrhizic acid (GA) against this toxicity using rat and human hepatocytes in monolayer culture. After a 48 h treatment, cocklebur as well as atractyloside induced concentration-dependent hepatotoxicity according to obvious decreases of cell viability, intracellular glutathione (GSH) content and albumin secretion in rat hepatocytes. Compared with rat hepatocytes, human hepatocytes seemed to be less sensitive to cocklebur-induced hepatotoxicity, indicative of species differences between humans and rats. Furthermore, as expected, GA showed significant protective effects on cocklebur hepatotoxicity in both human and rat hepatocytes. Overall, these results suggested that hepatocytes in vitro, particularly human hepatocytes, could be a useful tool for herbal hepatotoxicity screening in vitro.