Three-dimensional liver in vitro systems have recently attracted a lot of attention in drug development. These systems help to gain unprecedented insights into drug-induced liver injury (DILI), as they more closely reproduce liver biology, and as drug effects can be studied in isolated and controllable microenvironments. Many groups established human-based in vitro models but so far neglected the animal equivalent, although the availability of both models would be desirable. Animal in vitro models enable back- and forward translation of in vitro and in vivo findings, bridge the gap between rodent in vivo and human in vitro scenarios, and ultimately support the interpretation of data generated with preclinical species and humans. Since mice are often used in drug development and physiologically relevant in vitro systems are lacking, we established, for the first time, a mouse liver model that encompasses primary parenchymal and non-parenchymal cells with preserved viability and functionality over three weeks. Using our three-dimensional liver spheroids, we were able to predict the toxicity of known DILI compounds, demonstrated the interaction cascades between the different cell types and showed evidence of drug-induced steatosis and cholestasis. In summary, our mouse liver spheroids represent a valuable in vitro model that can be applied to study DILI findings, reported from mouse studies, and offers the potential to detect immune-mediated drug-induced liver toxicity.