The analysis of neurochemicals from the brain represents a challenge for current analytical techniques due to a variety of factors, such as compositional complexity, limited amounts of sample and endogenous inferences. Advances in mass spectrometry (MS) provide great opportunities for the sensitive measurement of neurochemicals, offering benefits including simple sample preparation, broad capability for analysis of diverse compounds and rich structural information of analytes. Until recently, however, limited numbers of studies have reported on the analysis of small molecular neurochemicals, such as classical neurotransmitters, in part due to the difficulties in separation of polar molecules by using current chromatography techniques with MS-compatible conditions. By contrast, MS has become an indispensable tool for neuropeptide analysis , offering tremendous potential in the discovery of novel signaling peptides and biomarkers. This review covers recent advances in MS-based neurochemical analysis , including a comparison with related detection techniques, chromatographic separation and neuropeptide discovery. Issues relating to in vivo sample collection and sample preparation are discussed. To provide a wider view of the capability of MS in basic neuroscience and clinical research, we discuss MS-based neurochemical analysis conducted in different animal models and humans. We specifically highlight perspectives for the use of MS for brain functional studies and drug discovery in nonhuman primates.