Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding with obvious reasons are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that impact the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible off-target effects. The NIH Clinical Collection (NCC) library containing 727 drugs was screened and 9 drugs, including the most commonly prescribed anticoagulant warfarin, were identified. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), while idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other three drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs targeting VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.