Cerebrospinal fluid (CSF) is a biofluid in direct contact with the brain and as such constitutes a sample of choice in neu-rological disorder research, including neurodegenerative dis-eases 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 fractionation of tryptic peptides to alle-viate the complexity of the biofluid. The resulting 24 frac-tions were analysed using liquid chromatography coupled to a high-resolution and high-accuracy timsTOF Pro mass spec-trometer. This state-of-the-art mass spectrometry-based pro-teomic workflow allowed the identification of 3'174 proteins in CSF. The dataset reported herein completes the pool of the most comprehensive human CSF proteomes obtained so far. An overview of the identified proteins is provided based on gene ontology annotation. Mass and tandem mass spectra are made available as a possible starting point for further studies exploring the human CSF proteome. (c) 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ )