Background and Purpose: Rivaroxaban as an oral anticoagulant is widely used for the prevention and treatment of thromboembolic disease. Previous studies revealed cytochrome P450 (CYP)–mediated metabolism of rivaroxaban mainly involves CYP2J2 and CYP3A4. Imatinib, sunitinib and gefitinib are three tyrosine kinase inhibitors (TKIs) that are extensively applied for anti-cancer therapy. Statistical research has shown cancer patients are at approximately 4-7–fold higher risk of vein thromboembolism than normal patients. Therefore, rivaroxaban and TKIs have a profound combination foundation. This study aimed to comprehensively assess the combination safety of rivaroxaban with TKIs in vitro. Experimental Approach: First, the inhibitory activity of the three TKIs was screened. Second, to comprehensively evaluate their inhibitory potential, the reversible and mechanism-dependent inhibitory kinetic constants of three TKIs on CYP2J2 and CYP3A4 were determined. Docking simulation was used to explore the molecular mechanism. Finally, drug-drug interaction (DDI) risks of the combination were assessed using pharmacokinetic data of cancer patients. Key Results: Imatinib and gefitinib exerted significant reversible inhibition of both CYP2J2 and CYP3A4, while sunitinib only showed reversible inhibition of CYP3A4, not CYP2J2. Three TKIs also showed time-dependent inactivation of CYP3A4 and slightly on CYP2J2. Notably, sunitinib had a significantly stronger inactivation effect on CYP3A4 than the other TKIs, with a 4.14-fold IC50 shift. Imatinib was predicted to cause a 114–244% increase in rivaroxaban exposure. Conclusion and Implication: Imatinib showed the strongest inhibition, which was predicted to have a moderate DDI risk. These results provide evidence for medication guidance when combining rivaroxaban with TKIs.