Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labelling approach. We herein describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labelling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis cohort. Two proteins, GAP43 and SERPINA3, were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in late stage secondary progressive multiple sclerosis compared to early stages of multiple sclerosis and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all multiple sclerosis patients compared to controls. The developed assay procedure now offers new possibilities for broad scale protein profiling of CSF within neurological disorders. This article is protected by copyright. All rights reserved.