Regulation of thrombin commands a high interest in the medical community because it is a key enzyme involved in blood coagulation, being an important target for preventing thrombotic disorders, such as venous thromboembolism and stroke. My thesis focuses on design, synthesis and characterization of new inhibitors of thrombin as potential new anticoagulants. In our work, we identified unsulfated small molecules and charge-reduced modified-heparin derivatives as novel allosteric inhibitors of thrombin.