Low levels of hypocretin-1 (Hcrt1) in cerebrospinal fluid (CSF) are associated with narcolepsy type 1 (NT1). Although immunoassays are prone to antibody batch differences, detection methods and variation between laboratories, the standard method for Hcrt1 measurement is a radioimmunoassay (RIA). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is an antibody- and radioactive free alternative for precise measurement of Hcrt1. We developed an LC-MS/MS method for measurement of Hcrt1 in CSF with automated sample preparation by solid-phase extraction (SPE). The LC-MS/MS method was compared with the RIA method for Hcrt1 detection. CSF samples from healthy subjects and NT1 patients was obtained by lumbar puncture. NT1 patients were diagnosed according to the minimal criteria by the International Classification of Sleep Disorders (ICSD). The LC-MS/MS method showed linearity across the range of calibrators and had a limit of detection (LOD) of 2.5 pg/mL and a limit of quantitation (LOQ) of 3.6 pg/mL. Comparison of the LC-MS/MS method with RIA revealed a 19 times lower level in healthy controls and 22 times lower level in NT1 patients with the LC-MS/MS method than with RIA. Bland-Altman analysis demonstrated agreement between the methods. These results question what is detected by RIA and strongly suggest that the physiological concentrations of the peptide are much lower than previously believed. LC-MS/MS proves to be an alternative for detection of Hcrt1 for diagnosis of narcolepsy.