Objective: This study aimed to examine the interspecies differences in CYP enzyme activity and mRNA expression between canine and human hepatocytes following treatment with acetaminophen (AAP), diclofenac (Dic), or valproic acid (VPA). Methods: We determined the 24-h exposure half-maximal inhibitory concentration (IC 50 ) values of AAP, Dic, and VPA in canine and human hepatocytes. Based on these IC 50 concentrations, we compared drug-induced alterations in various parameters, including immunocytochemistry, transcriptomic profiles (RNA-seq), and CYP activity, to assess changes at the gene and protein levels. Results: AAP and VPA increased CYP2J2 mRNA expression by 4.7- and 7.76-fold, respectively, whereas Dic increased CYP1A1 mRNA expression by 14.25-fold in canine hepatocytes. AAP, VPA, and Dic decreased CYP26B1 mRNA expression in canine hepatocytes by 0.03-, 0.12-, and 0.17-fold, respectively. Dic and VPA increased CYP1A1 mRNA expression by 5.53- and 6.66-fold, respectively, whereas AAP, VPA, and Dic decreased CYP4F22 mRNA expression by 0.03-, 0.13-, and 0.13-fold, respectively, in human hepatocytes. Conclusions and Relevance: The observed differences between species in CYP activity and mRNA levels in response to drug exposure highlight the importance of accurate and precise experimental models for the development of new medications.