Vinblastine (VBL) is a vinca alkaloid-class cytotoxic chemotherapeutic that causes microtubule disruption and is typically used to treat hematologic malignancies. VBL is characterized by a narrow therapeutic index, with key dose-limiting toxicities being myelosuppression and neurotoxicity. Pharmacokinetics (PK) of VBL is primarily driven by ABCB1-mediated efflux and CYP3A4 metabolism, creating potential for drug-drug interaction. To characterize sources of variability in VBL PK, we developed a physiologically based pharmacokinetic (PBPK) model in Mdr1a/b(-/-) knockout and wild-type mice by incorporating key drivers of PK, including ABCB1 efflux, CYP3A4 metabolism, and tissue-specific tubulin binding, and scaled this model to accurately simulate VBL PK in humans and pet dogs. To investigate the capability of the model to capture interindividual variability in clinical data, virtual populations of humans and pet dogs were generated through Monte Carlo simulation of physiologic and biochemical parameters and compared to the clinical PK data. This model provides a foundation for predictive modeling of VBL PK. The base PBPK model can be further improved with supplemental experimental data identifying drug-drug interactions, ABCB1 polymorphisms and expression, and other sources of physiologic or metabolic variability.