Overall, our results demonstrate that (i) various antibiotics reported to cause cholestasis and hepatocellular injury in the clinic can also induce such effects in in vitro human hepatocytes; (ii) PRAs cause the strongest cholestatic effects in the absence of cytotoxicity; (iii) cholestatic features occur early through ER stress; (iv) cytotoxic lesions are observed later through ER stress-mediated ROS generation; and (v) activation of the HSP27-PI3K-AKT pathway protects from cytotoxic damage induced by PRAs only.