Cerebrospinal fluid (CSF) contains peptides and proteins important for brain physiology and potentially also relevant to brain pathology. Therefore, CSF provides an attractive source for biomarker discovery in brain and neurological diseases. CSF proteomics provides an analytical challenge as ∼80% of proteins originate from serum, and removal of these major proteins is necessary to study brain-derived proteins that are present at low concentrations. In this book chapter, we describe a CSF sample pretreatment method to allow for in-depth and comparative analysis of the CSF proteome. To this end, we provide a protocol for batch-mode abundant protein depletion using the multiple affinity removal system MARS cartridge (Agilent) as well as for subsequent protein concentration using ultrafiltration with a 3 kDa molecular weight cutoff (MWCO) spin filter (Millipore). In addition, our experience with this depletion method coupled to 1D page/LC-MS/MS as well as other depletion and protein fractionation methods for CSF analysis is discussed.