Glial fibrillary acidic protein (GFAP) and protein S-100B are established indicators of astrogliosis in neuropathology. As GFAP and S-100B are expressed in different cell populations, variable cerebrospinal fluid (CSF) concentrations of these proteins might reflect disease-specific pathological profiles. Therefore we investigated CSF of patients with Alzheimer's disease (AD), patients with Creutzfeldt-Jakob disease (CJD), and non-demented control patients (CON). Measurement of GFAP and S-100B in CSF was performed by commercially available ELISA. Our results show that, in AD, there are significantly higher levels of GFAP concentrations, compared to CON (p = 0.001) and CJD patients (p = 0.009), whereas S-100B is much higher in CJD, compared to AD (p = 0.001) and CON (p = 0.001). In conclusion, GFAP and S-100B represent astroglial markers and the different levels of these proteins in CSF of AD and CJD patients might point to a distinct pathophysiological involvement in these diseases. Apart from pathophysiological aspects, GFAP in particular might serve as an additional diagnostic tool for AD, due to the fact that this protein does not correlate to established markers like tau and amyloid-beta such that analysis of GFAP may be useful for further differential diagnostic approaches in neurodegenerative diseases.