The liver is the primary organ involved in drug detoxification, hence being highly exposed to the risk of drug induced liver injury (DILI). DILI is the main cause for acute liver failure in western countries and post-marketing withdrawal of drugs. This is highly due to the poor identification of potential human hepatotoxins during the preclinical phase of drug discovery. It is not surprising that interspecies difference between animal models could generate diverse biological responses compared to the human liver. Similarly, the current-state-of-the-art human in vitro hepatic systems lack the cellular spatial arrangement and hepatocyte functionality observed in vivo. Identification of an in vitro hepatic system capable to reproduce the essential features of the human liver in vivo is thus beneficial to better understand DILI and how it is triggered. Historically, maintenance of primary hepatocyte in vitro has been challenging due the intrinsic finite proliferative capacity of these cells, the shortage of donor livers, and the inability to maintain their in vivo structural and functional identity in vitro. All of the above hurdles have been addressed with the advent of 3D epithelial structures called organoids, yet its use in the liver safety field is currently unexplored. Liver organoids have the great advantage to be highly proliferative, to maintain the identity of the tissue of origin, and to retain the bipotential ability to differentiate into hepatocyte organoids. In this Thesis, we generated an enhanced hepatocyte organoid system through the directed stepwise differentiation in vitro of progenitor liver organoids using chemically defined culture conditions. Validation of these organoids in the context of DILI, showed structural and functional formation of tubular like structures called bile canaliculi which are essential morphological structures for the hepatocytes to secrete drugs/metabolites and bile from the intracellular compartment. Drug distribution mapping analysis revealed the ability of these organoids to uptake exogenous compounds. Therefore, we examined the potentiality of the hepatocyte organoids to detect known human hepatotoxins, which resulted in the obstruction of hepatic bile clearance and downregulation of transmembrane proteins in a process called cholestasis. Furthermore, formation of typical features of drug-induced steatosis were observed upon treatment with steatotic drugs. In summary, our findings highlight how this novel differentiated organoid system could be a relevant human in vitro system with the potential to be incorporated into an initial drug safety screening, either as a replacement or complementary hepatic system.