BACKGROUND: Adrenomedullin (ADM) is a potent hormone-like peptide rapidly induced by hypoxia and inflammatory cytokines in the early stages of sepsis. For this reason, the dosage of its more stable precursor fragment called mid-regional (MR)-proADM is currently recommended to assist in triaging patients in the emergency department. Since MR-proADM dosage is currently only approved for use in plasma, we validated its dosage in cerebrospinal fluid (CSF) samples to improve the diagnosis of central nervous system (CNS) diseases. METHODS: MR-proADM concentrations were measured in samples using a fully automated platform (Brahms Kryptor Gold Analyzer, Thermo Scientific, Germany), applying the same analytical conditions in plasma and CSF samples, to finally set up an accurate laboratory protocol to validate its dosage in CSF. RESULTS: MR-proADM is highly stable in CSF samples stored at room temperature for up to 48 h, allowing it to be measured with confidence also in CSF samples that may be left on the bench for several hours. In addition, the repeatability and within-laboratory precision of the MR-proADM assay using CSF samples appeared equal to or better than those obtained by the manufacturer using plasma samples, allowing the use of this assay, with high precision, also for CSF samples. CONCLUSION: The reliable measure of MR-proADM in CSF and the role of this molecule in CNS will allow its introduction in the diagnostic process of infectious, inflammatory, and degenerative neurological diseases.