Abstract: Lipids are essential functional biomolecules within cells, characterized by their abundant quantities, diverse species, and versatile structures. In recent years, numerous scholars have devoted efforts to developing advanced techniques for the accurate characterization of lipid fine structures, aiming to identify novel lipid molecules as disease biomarkers and for lipid functional research. Lipid reference materials play important roles in the qualitative and quantitative analysis of lipids, but due to the diversity of lipid fine structures, their preparation and quantification are challenging tasks. Mass spectrometry (MS), renowned for its robust capability in accurate compound structure measurement, has gained significant attention in the field of lipid fine structure characterization. MS-based analytical methods for lipid fine structure analysis have rapidly progressed, offering a novel perspective for lipid study, deepening our understanding of lipid structural diversity and the lipid metabolic network. Moreover, these methods have propelled the screening of potential disease biomarkers and the discovery of new metabolic pathways. Currently, MS-based analytical methods for accurate lipid fine structure measurement primarily employ two strategies: novel ion activation/dissociation methods and specific chemical derivatization. These approaches demonstrate excellent analytical performance for a variety of lipid fine structure isomers. This review summarizes the MS-based analytical methods for lipid fine structure and provides an outlook on their applications in the preparation of lipid reference materials.