Cerebrospinal fluid (CSF) is an extremely useful matrix for biomarker research for several purposes, such as diagnosis, prognosis, monitoring, and identification of prominent leads in pathways of neurologic diseases. Such biomarkers can be identified based on a priori hypotheses around prominent protein changes, but also by applying -omics technologies. Proteomics is widely used, but metabolomics and transcriptomics are rapidly revealing their potential for CSF studies. The basis of such studies is the availability of high-quality biobanks. Furthermore, profound knowledge and consequent optimization of all aspects in biomarker development are needed. Here we discuss current knowledge and recently developed protocols for successful biomarker studies, from collection of CSF by lumbar puncture, processing, and biobanking protocols, preanalytic confounding factors, and cost-efficient development and validation of assays for implementation into clinical practice or research.