Although the diagnosis of multiple sclerosis (MS) may be clinically suspect and the magnetic resonance imaging findings compatible, cerebrospinal fluid (CSF) analysis remains mandatory in order to support the diagnosis. This is especially important since our understanding of the defining disease pathogenesis remains incomplete. However, there is no specifically diagnostic CSF test. And until recently, laboratory techniques for CSF analysis had not been rigorously standardized. Unconcentrated CSF without fixative should be used for the determinations of cell count and differential, protein and glucose, lactate, myelin basic protein, and the CSF/serum albumin ratio which is an indicator of blood-CSF barrier disruption. Additionally, CSF immunoglobulin-gamma (IgG) determinations are of major importance and are now included in the MS diagnostic criteria. Testing for oligoclonal IgG bands utilizing isoelectric focusing with IgG immunoblotting, the IgG synthesis rate, and the IgG index should be included. CSF analysis for kappa light chains and IGM may be diagnostically helpful. The search for biomarkers including those possibly present in the CSF which could predict and assess the course as well as response to treatment in a particular MS patient has not yet been successful. CSF immunoglobulin and T-cell/B-cell patterns, soluble HLA class I and II antigens, nitrous oxide metabolites, neurofilament and microtubule components and antibodies, tau protein, 14-3-3-protein, neuronal cell and intercellular adhesion molecules, and chemokines are actively being investigated as MS biomarkers.