Most central nervous system (CNS) disorders are currently incurable due to our limited understanding of disease mechanisms and the challenges of CNS drug development. Mass Spectrometry (MS) has become one of the foremost technologies to analyze diverse biomolecules with unbeaten throughput and accuracy to contribute to our fundamental understanding of the CNS. MS-based techniques are also widely applied to decipher the molecular mechanisms of neurological disorders and discover biomarkers and therapeutic targets for brain diseases. This Method Collection will highlight recent advancements and methods in various MS techniques and MS-based analyses of diverse biomolecules, including but not limited to MS-based proteomics, peptidomics, metabolomics, lipidomics, MS-imaging, single-cell analysis, and biomarker discovery, with a special focus in studying the CNS. MS-based methods to analyze samples from different cellular and animal models of neurological disorders, as well as human patient samples, will also be included in this collection. The goal is to provide video-based protocols and promote best practices in cutting-edge MS-based methodologies, assisting the researchers in the field to advance new technologies and deepen our understandings of the CNS.