Cholestasis represents a major subtype of drug-induced liver injury and novel preclinical models for its prediction are needed. Here we used primary human hepatocytes (PHH) from different donors in 2D-sandwich (2D-sw) and/or 3D-spheroid cultures to study inter-individual differences in the response towards cholestatic hepatotoxins after short-term (48–72 hours) and long-term repeated exposures (14 days). The cholestatic liabilities of drugs were determined by comparing cell viability upon exposure to the highest non-cytotoxic drug concentration in the presence and absence of a non-cytotoxic concentrated bile acid mixture. In 2D-sw culture, cyclosporine A and amiodarone presented clear cholestatic liabilities in all four PHH donors tested, whereas differences in the susceptibility of the various PHH donors towards the cholestatic toxicity of bosentan, chlorpromazine and troglitazone were observed. In PHH from one donor, the cholestatic liabilities of chlorpromazine and troglitazone could only be detected after long-term repeated exposures when maintained in 3D-spheroid culture, but not after short-term exposures in either 2D-sw or 3D-spheroid culture, suggesting that cholestatic hepatotoxicity may require time to develop. In conclusion, inter-individual susceptibility exists towards drug-induced cholestasis, which depends on the compound as well as the exposure time.