Although neuroimaging is useful in differentiating ischemic (IS) from hemorrhagic (ICH) stroke in the Emergency Department, a wide-available rapid biochemical test would add advantages in the pre-hospital triage and management of stroke patients. Our aim was to examine the predictive value of a panel of blood-borne biomarkers to differentiate IS from ICH. Admission blood samples obtained within 24. h from stroke symptoms onset were tested by ELISA for CRP, D-dimer, sRAGE, MMP9, S100B, BNP, NT-3, caspase-3, chimerin-II, secretagogin, cerebellin and NPY. The complete protocol was achieved in 915 patients (776 IS, 139 ICH). Among blood samples obtained < 6. h from symptoms onset (n = 337), S100B levels were increased in ICH (107.58 vs 58.70 pg/mL; p < 0.001) whereas sRAGE levels were decreased (0.77 vs 1.02. ng/mL; p = 0.009) as compared to IS. In this subset of patients S100B (OR 3.97 95% CI 1.82-8.68; p = 0.001) and sRAGE (OR 0.22 95% CI 0.10-0.52; p < 0.001) were independently associated with ICH. A regression tree was created by CART method showing good classification ability (AUC = 0.762). Similar results were found for samples obtained within 3. h. In conclusion, a combination of biomarkers including those of the S100B/RAGE pathway seems promising to achieve a rapid biochemical diagnosis of IS versus ICH in the first hours from symptoms onset. This article is part of a Special Issue entitled: Translational Proteomics. © 2012.