<p>Cigarette smoke contains compounds that significantly affect drug interactions in clinical settings, primarily through the induction of cytochrome P450 (CYP) enzymes. Given the complexity of cigarette smoke, use of cigarette smoke extract (CSE) in in vitro systems is essential for mimicking in vivo effects and analyzing CYP enzyme induction. Regulatory authorities recommend testing drug–drug interactions; however, there is currently no in vitro model to study smoke–drug interactions. This study developed a model using primary human hepatocytes (PHH) to analyze CYP enzyme induction by CSE inspired by the ICH M12 (2024) guideline. The robustness of the model was evaluated by analyzing the effects of CSE derived from three lots of research cigarettes on three PHH donors. The parameters of CSE production were optimized for a 2.5-min cigarette burning and a cell culture medium as CSE solvent. A reliable, concentration-dependent induction of CYP1A1 and CYP1A2 by CSE across different donors and cigarettes was verified based on an enzyme activity (LC–MS/MS) and mRNA expression levels (qRT-PCR). The strongest induction was observed for CYP1A1 mRNA with at least 15-fold, and CYP1A1 enzyme activity with at least 57-fold induction, across all donors. Protein levels of induced CYP1A1 in PHH were comparable to those of non-induced CYP1A2. Therefore, the data suggest a notable role of induced CYP1A1 in liver metabolism. Additionally, CSE induced the mRNA expression levels of CYP2B6, CYP2C8, and CYP3A4. This model has the potential to provide in vitro smoke–drug interaction results prior to clinical trials and can therefore assist in clinical study planning.</p>