Updated publication reference for PubMed record(s): 32478154. Cerebrospinal fluid (CSF) is a biofluid in direct contact with the brain and as such constitutes a sample of choice in the study of neurological disorders, including neurodegenerative diseases such as Alzheimer or Parkinson. Human CSF has still been less studied using proteomic technologies compared to other biological fluids such as blood plasma or serum. In this work, a pool of “normal” human CSF samples was analysed using a shotgun proteomic workflow that combined removal of highly abundant proteins by immunoaffinity depletion and isoelectric focussing electrophoresis fractionation of tryptic peptides to alleviate the complexity of the biofluid. The resulting 24 fractions were analysed using liquid chromatography coupled to a high-resolution and high-accuracy timsTOF Pro mass spectrometry instrument. This state-of-the-art protocol allowed the identification of 3’174 proteins in CSF.