Cerebrospinal fluid (CSF) is in direct contact with the brain and serves as a valuable specimen to examine neurodegenerative diseases in order to identify suitable diagnostic biomarkers. In the field of neuroproteomics the most popular mass spectrometry-based strategy for biomarker discovery studies is data-dependent acquisition (DDA). Drawbacks of this method are stochastic and irreproducible precursor ion selection. Recently, data-independent acquisition (DIA) emerged as an alternative method. In this study we evaluate the use of DIA for native CSF analytics with particular focus on spectral library generation for data analysis, DIA data reproducibility and identification of brain specific proteins.