Enzymatic reactions usually occur with a high substrate, product, or substrate–product stereoselectivity. In many instances, information on the stereochemical disposition of biologically active metabolites is indispensable for a complete understanding of biological processes and metabolic states. This raised interest in structural isomerism and stereoisomerism in metabolomics and lipidomics. While structural and geometrical isomers, as well as diastereomers, can be principally distinguished by achiral liquid chromatography (LC) and also ion-mobility mass spectrometry (MS), enantiomers require enantioselective separation methods. While untargeted enantioselective metabolomics is mostly based on the derivatization of metabolites with a chiral derivatizing agent, followed by LC separation on achiral columns and hyphenation with high-resolution mass spectrometry, targeted enantioselective LC with chiral stationary phases mostly employs robust triple quadrupole (QqQ) mass spectrometry for detection. It is a powerful technology, yet it focuses primarily on a broad, metabolite class–wide coverage. This chapter describes four distinct fields of application of targeted enantioselective LC–tandem mass spectrometry assays for important and representative metabolite classes. The first one deals with the enantioselective analysis of oxylipins using polysaccharide-based chiral columns, with protocols applicable for platelets and plasma; the second focuses on 3-hydroxy fatty acid enantiomer separations in plasma and platelets; the third application provides two workflows for proteinogenic enantioselective amino acid analysis; and the fourth application proposes methodologies for enantioselective short branched-chain fatty acid analysis. Critical aspects are discussed.