Cannabidiol (CBD) and valproate (VPA) are anti-epileptic medications commonly co-prescribed to treat seizures due to Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex in children. Clinical trial data have demonstrated that CBD carries a risk for severe hepatotoxicity that is greatly increased when prescribed with VPA through an unknown mechanism. The aim of this study was to investigate CBD-induced liver injury in combination with VPA using an in vitro liver model. Three-dimensional human hepatocyte spheroids are an emerging in vitro system that allows investigation of long-term toxicity. Spheroids derived from primary human hepatocytes were treated with vehicle control, 2-200 μM CBD, 0.5-20 mM VPA, CBD + VPA, and 0.1-10 mM acetaminophen (positive control). After 24 h, 8 days, and 15 days of exposure, spheroids were analyzed for ATP depletion, urea production, and CBD and VPA metabolite generation. Untargeted metabolomic analysis was also conducted. A delayed-onset, dose-dependent hepatotoxicity was observed in spheroids exposed to each drug treatment compared to vehicle control. This study is the first to recapitulate the hepatotoxic drug interaction of CBD and VPA in vitro and demonstrates the utility of human hepatocyte spheroids for toxicity studies. Future work is needed to examine mechanisms of CBD-induced hepatotoxicity with VPA.