Polypharmacy-related drug-drug interactions (DDIs) are a significant and growing healthcare concern. An increasing number of therapeutic drugs on the market underscores the necessity to accurately assess new drug combinations during preclinical evaluation for DDIs. In vitro primary human hepatocytes (PHH) models are only applicable for short-term induction studies because of their rapid loss of metabolic function. Though coculturing nonhuman stromal cells with PHH has been shown to stabilize metabolic activity long-term, there are concerns about human specificity for accurate clinical assessment. In this study, we demonstrated a PHH-only liver microphysiological system in the Liver Tissue Chip is capable of maintaining long-term functional and metabolic activity of PHH from 3 individual donors and thus a suitable platform for long-term DDI induction studies. The responses to rifampicin induction of 3 PHH donors were assessed using cytochrome P450 activity and mRNA changes. Additionally, victim pharmacokinetic studies were conducted with midazolam (high clearance) and alprazolam (low clearance) following perpetrator drug treatment, rifampicin-mediated induction, which resulted in a 2-fold and a 2.6-fold increase in midazolam and alprazolam intrinsic clearance values, respectively, compared with the untreated liver microphysiological system. We also investigated the induction effects of different dosing regimens of the perpetrator drug (rifampicin) on cytochrome P450 activity levels, showing minimal variation in the intrinsic clearance of the victim drug (midazolam). This study illustrates the utility of the Liver Tissue Chip for in vitro liver-specific DDI induction studies, providing a translational experimental system to predict clinical clearance values of both perpetrator and victim drugs. SIGNIFICANCE STATEMENT: This study demonstrated the utility of the Liver Tissue Chip with a primary human hepatocyte-only liver microphysiological system for drug-drug interaction induction studies. This unique in vitro system with continuous recirculation maintains long-term functionality and metabolic activity for up to 4 weeks, enabling the study of perpetrator and victim drug pharmacokinetics, quantification of drug-induced cytochrome P450 mRNA and activity levels, investigation of patient variability, and ultimately clinical predictions.