With the current advancement in mass spectrometry (MS)-based lipidomics, the knowledge of lipidomes and their diverse roles has greatly increased, enabling a deeper understanding of the action of bioactive lipid molecules in plant- and animal-based foods. This review provides in-depth information on the practical use of MS techniques in lipidomics, including lipid extraction, adduct formation, MS analysis, data processing, statistical analysis, and bioinformatics. Moreover, this contribution demonstrates the effectiveness of MS-based lipidomics for identifying and quantifying diverse lipid species, especially triacylglycerols and phospholipids, in foods. Further, it summarizes the wide applications of MS-based lipidomics in food science, such as for assessing food processing methods, detecting food adulteration, and measuring lipid oxidation in foods. Thus, MS-based lipidomics may be a useful method for identifying the action of individual lipid species in foods.