The invention relates to an in vitro immunoassay for quantifying thrombin in a sample comprising anti-thrombin III (AT-III) and thrombin. The method comprises the following steps: contacting the sample with a small molecule that recognizes the substrate binding site of thrombin; contacting the thrombin with a thrombin specific antibody raised against a thrombin blocked in the active site; and measuring the level of bound antibody.