Arterial thrombosis causes heart attacks and strokes and is the leading cause of morbidity and mortality in the United States. However, there are only a few adequate therapies available for treating arterial thrombosis. Thus, there is a clear need for new approaches and new targets in the prevention and treatment of arterial thrombosis. Protein disulfide isomerase (PDI) is expressed on vascular cells following injury and has been shown to be a critical regulator of thrombus formation in vivo. Since inhibition of PDI prevents platelet accumulation and fibrin generation, it makes it a tractable target for the development of new antithrombotics. A high throughput screen (HTS) was conducted to identify potent and selective inhibitors of PDI. An insulin-based turbidometric assay was used to screen 348,505 compounds of the MLSMR library. Potential PDI inhibitors were further characterized via additional specificity and cytotoxicity assays. We report the identification and characterization of a small-molecule probe (ML359) shown to be a specific inhibitor of PDI. ML359 shows no cytotoxicity in three human cell lines, and some activity in inhibiting platelet aggregation in vitro. ML359 will provide more understanding of the mechanisms of PDI in platelet functions. It also could lead to the development of a new class of antithrombotics that could establish PDI as suitable drug target.