This chapter describes the methods for pre-analytical processing and biobanking of cerebrospinal fluid (CSF) samples. There is increasing knowledge that pre-analytical procedures and storage conditions can influence biomarker results, which is relevant to all kinds of fluids and thus also biomarkers measured in CSF. The pre-analytical phase includes patient-related factors, such as fasting, as well as processing factors, such as delay between collection and processing, type of tube, or number of transfers of the fluid. As for the processing factors, a distinction can be made whether analysis is performed for routine care or research purposes, which mostly affects the storage time and storage temperatures. For example, routine analysis usually implies storage of days to weeks at −20 °C or 4 °C, while research analysis usually implies storage for years at −80 °C. The presented protocol is based on previously published protocols for CSF collection and long-term biobanking in the context of biomarker development or research, with adaptations based on experimental evidence collected over time.