Alisporivir is a novel cyclophilin-binding molecule with potent anti-hepatitis C virus (HCV) activity. In vitro data from human liver microsomes suggest that alisporivir is a substrate and a time-dependent inhibitor (TDI) of CYP3A4. The aim of the current work was to develop a novel physiologically based pharmacokinetic (PBPK) model to quantitatively assess the magnitude of CYP3A4 mediated drug-drug interactions with alisporivir as the substrate or victim drug. Towards that, a Simcyp PBPK model was developed by integrating in vitro data with in vivo clinicalfindings to characterize the clinical pharmacokinetics of alisporivir and further assess the magnitude of drug-drug interactions. Incorporated with absorption, distribution, elimination, and TDI data, the model accurately predicted AUC, Cmax, and tmax values after single or multiple doses of alisporivir with a prediction deviation within ± 32%. The model predicted an alisporivir AUC increase by 9.4-fold and a decrease by 86% when alisporivir was co-administrated with ketoconazole (CYP3A4 inhibitor) or rifampin (CYP3A4 inducer), respectively. Predictions were within ± 20% of the observed changes. In conclusion, the PBPK model successfully predicted the alisporivir PK and the magnitude of drug-drug interactions.