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Acute stroke management is critically time-sensitive and challenging. Blood-based biomarkers that can differentiate acute ischemic stroke (IS) from hemorrhagic stroke (HS) can greatly facilitate triage and early management. Admission blood samples obtained within 6 h of stroke symptom onset were analyzed in a derivation/validation design. GFAP, N-FL, NT-proBNP, copeptin, neutrophils (%), NLR, and platelet counts were assessed in the derivation cohort. The informative markers and the derived cutoff values were evaluated in the validation cohort. GFAP > 703 pg/mL showed a PPV of 76.9% and NPV of 95.8% for differentiating HS from IS. Multiple logistic regression analysis showed that GFAP and NT-proBNP were independent variables associated with IS and HS differentiation. Furthermore, applying a combined cutoff (GFAP > 703 pg/mL and NT-proBNP ≤ 125 pg/mL) for HS detection increased the PPV in both the derivation and validation cohorts (93.3% and 100%, respectively). GFAP and NT-proBNP levels were validated as informative blood biomarkers in the differentiation of IS and HS and using a combination of GFAP and NT-proBNP is suggested as a feasible strategy to differentiate stroke subtypes in the hyperacute phase of stroke. [ABSTRACT FROM AUTHOR]