<b><i>Background:</i></b> Both, acute ischemic stroke (AIS) and hemorrhage stroke (intracerebral hemorrhage, ICH) require early attention but different treatment strategies. Plasma glial fibrillary acidic protein (GFAP) levels were found to be elevated in ICH patients after they arrived in the hospital. Because treatment options differed, we sought to determine whether GFAP can be used to accurately differentiate between of AIS and ICH in the prehospital setting. <b><i>Methods:</i></b> We assessed acute stroke patients in the Stroke Emergency Mobile (STEMO). STEMO is a stroke ambulance staffed by a specialized team including a neurologist and equipped with a computed tomography scanner plus a point-of-care laboratory. The STEMO ambulance is integrated in the emergency medical system of Berlin, Germany. Following prehospital stroke diagnosis, blood was drawn and subsequently analysed using research assays from Roche diagnostics. The clinical accuracy of plasma GFAP was tested using a cut-off value of 0.29 ng/ml. <b><i>Results:</i></b> Blood samples of 74 patients were analysed. Twenty-five patients had ICH (mean age 69 ± 11 years, median National Institutes of Health Stroke Scale (NIHSS) 15) and 49 IS (mean age 75 ± 10 years, median NIHSS 6). Nine ICH (0 IS patients) had GFAP-levels above 0.29 ng/ml. The sensitivity and specificity of GFAP for differentiating between ICH and AIS were 36.0 and 100%. The sensitivity for ICH volume >15 ml was 61.5%. ICH patients without GFAP elevation had significantly smaller hemorrhage volumes (median 4.5 vs. 37.6 ml, p = 0.004) and were less likely to deteriorate (19 vs. 56%, p = 0.087). <b><i>Conclusions:</i></b> GFAP levels >0.29 ng/ml were seen only in ICH, thus confirming the diagnosis of ICH during prehospital care. However, sensitivity is low particularly in smaller hemorrhages.