AbstractLipids are amongst the most important organic compounds in living organisms, where they serve as building blocks for cellular membranes as well as energy storage and signaling molecules. Lipidomics is the science of the large-scale determination of individual lipid species, and the underlying analytical technology that is used to identify and quantify the lipidome is generally mass spectrometry (MS). This review article provides an overview of the crucial steps in MS-based lipidomics workflows, including sample preparation, either liquid–liquid or solid-phase extraction, derivatization, chromatography, ion-mobility spectrometry, MS, and data processing by various software packages. The associated concepts are discussed from a technical perspective as well as in terms of their application. Furthermore, this article sheds light on recent advances in the technology used in this field and its current limitations. Particular emphasis is placed on data quality assurance and adequate data reporting; some of the most common pitfalls in lipidomics are discussed, along with how to circumvent them.