Recent studies have evaluated proper acquisition and storage procedures for the use of serum or plasma for mass spectrometry (MS)-based proteomics. The present study examines the proteome stability of human cerebrospinal fluid (CSF) over time at 23°C (room temperature) and 4°C using surface-enhanced laser desorption/ionization time-of-flight MS. Data analysis revealed that statistically significant differences in protein profiles are apparent within 4 h at 23°C and between 6 and 8 h at 4°C. Inclusion of protease and phosphatase inhibitor cocktails into the CSF samples failed to significantly reduce proteome alterations over time. We conclude that MS-based proteomic analysis of CSF requires careful assessment of sample collection procedures for rapid and optimal sample acquisition and storage.