Thrombin, a central factor in thrombogenesis, affects cells in the brain through protease activated receptors. Low levels of thrombin activity are neuroprotective while higher levels are deleterious, and we have therefore developed a new method for its direct quantitative measurement in brain slices following stroke. Thrombin activity was measured by a fluorescent substrate on fresh coronal slices taken from the ipsilateral and contralateral hemispheres 24-72 h following permanent right middle cerebral artery occlusion. Prolyl endopeptidase and aminopeptidases were inhibited as a critical step to insure the specificity of the assay for thrombin detection. Infarct volume was assessed using TTC staining. Thrombin activity in the right ischemic hemisphere was significantly higher compared to the contralateral hemisphere (32 ± 6 and 27 ± 10 mU/ml, mean ± SE in the two most affected slices from the ischemic hemisphere vs. 21 ± 6 and 8 ± 2 mU/ml in corresponding contralateral slices; p < 0.05). Thrombin levels in the ischemic and contralateral hemispheres were significantly higher compared to healthy control mice and were above the range known to be protective to brain cells. A significant correlation was found between thrombin activity in the ischemic hemisphere and the infarct volume. Results of studies based on this method may translate into potential thrombin based therapies.