For in vitro studies that predict the safety of systemically circulating drug metabolites, evaluating the formation and biliary excretion of hepatic drug metabolites in human hepatocytes is necessary. However, conventional sandwich-cultured hepatocytes (SCH) form closed bile canaliculi that preclude direct collection of excreted biliary metabolites. Therefore, this study assessed whether primary cultured hepatocytes with an induced open-form bile canaliculus (icHep) provide a suitable system for evaluating the partitioning of drug metabolites produced in hepatocytes into the bile-side and blood-side chambers. Hepatocytes derived from human liver chimeric mice were cultured on claudin-1-coated plates, which facilitated the formation of an open bile canalicular lumen, as confirmed by co-immunostaining for multidrug resistance-associated protein 2 and zonula occludens 1. RNA-sequencing analysis revealed that the expression of representative pharmacokinetic-related genes in the icHep system were comparable with those in conventional SCH. Following the addition of the parent drugs to the blood-side chamber of the permeation assay, metabolites were successfully collected from both the blood- and bile-side chambers. Ziprasidone sulfoxide and mycophenolic acid glucuronide were predominantly recovered from blood-side chamber, whereas celecoxib carboxylic acid and SN-38 glucuronide were preferentially excreted into the bile-side chamber. The partitioning of metabolites observed in the icHep system was consistent with human in vivo data. Overall, the icHep system enables evaluation of drug metabolite partitioning into the bile- and blood-side. Hence, this system represents a physiologically relevant in vitro system for human pharmacokinetic studies.