In this chapter the state-of-the-art in enantioselective metabolomics focusing on polar metabolites analyzed by liquid chromatography is summarized. The different methodologies commonly employed are outlined and critically discussed. Nowadays, direct enantioselective metabolomics can make use of several modern chiral HPLC as well as, recently, UHPLC columns containing chiral stationary phases based on various selectors, such as polysaccharide derivatives, macrocyclic antibiotics, chiral crown ethers, chiral ion exchangers, donor–acceptor phases and others. Where metabolites are concerned, many of them show class specific application profiles (e.g. for amino acids), such as chiral crown ether CSP, zwitterionic chiral ion-exchangers and teicoplanin CSP, or even wider scopes of applicability, such as amylose and cellulose tris (3,5-dimethylphenylcarbamate) CSPs. Since mass spectrometry is the detection principle of first choice in metabolomics applications, research concentrates on analytical separation systems that are compatible with MS detection. Since enantioselective UHPLC columns have only become available recently and often require tagging of the polar metabolites, indirect approaches have been utilized frequently up to now in particular for untargeted enantioselective metabolomics. A significant number of chiral derivatizing agents have been described for this purpose. Some caveats have to be considered to obtain accurate enantioselective assays by the indirect approach and are discussed in this chapter. The integration of these concepts in targeted and untargeted workflows is described, and some illustrative examples are given for targeted and untargeted enantioselective metabolomics applications.