Background <p>S100β protein has interesting metrological characteristics in the general population with mild traumatic brain injury (TBI) but its value in the subpopulation of patients on antithrombotic drugs (ATD) remains poorly documented.</p> Objective <p>The main objective was to evaluate the performance of S100β assay in ruling-out the presence of a cerebral injury after mTBI in subjects on ATD.</p> Design, settings and participants <p>This prospective single-center study was conducted between March 2020 and September 2021 in the Emergency Department of a French hospital. Patients on ATD consulting for mTBI having occurred less than 6&#xa0;h previously were included.</p> Outcomes measure and analysis <p>The S100β assay was to have been performed within 6&#xa0;h of the TBI, and a cut-off value of 0.105&#xa0;µg/L was applied. The standard diagnosis was provided by cerebral computed tomography (CCT). The primary endpoint was the metrological qualities of S100β for detecting intracranial complication following TBI. Economic impact was evaluated as a secondary endpoint.</p> Results <p>245 patients were included, with a mean age of 81.5 (± 11.8) years. CCT showed cerebral injury for 15 (6.1%) patients. The S100β assay was negative for 58 (23.7%) patients. Sensitivity was 100% (95% CI: 78.2% − 100%), specificity 25.2% (95% CI: 19.7% − 31.3%), and NPV 100% (95% CI: 93.8% − 100%). The S100β assay could have avoided CCT for 23.7% of the subjects, which would save €82.03 per patient compared to the current strategy.</p> Conclusion <p>The use of S100β within 6&#xa0;h of a TBI among subjects on ATD is efficacious in safely discounting cerebral injury, with an appreciable economic saving.</p> Clinical trial registration <p>NCT04305821 (2020-03-09)</p>