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Metabolomics is a rapidly emerging field of functional genomics research whose aim is the comprehensive analysis of low molecular weight metabolites in a biological sample. Capillary electrophoresis–electrospray ionization-mass spectrometry (CE–ESI-MS) represents a promising hyphenated microseparation platform in metabolomics, since a majority of primary metabolites are intrinsically polar. CE–ESI-MS offers a convenient format for the separation of complex mixtures of cationic, anionic, and/or zwitterionic metabolites, as well as their isobaric/isomeric ions without complicated sample handling. Moreover, online sample preconcentration with desalting is readily integrated during separation prior to ionization, where the migration behavior and ionization response of metabolites can be predicted based on their fundamental physicochemical properties. Herein, we describe recent developments in CE–ESI-MS with emphasis on practical protocols necessary for realizing reliable analyses as applied to targeted metabolite profiling and untargeted metabolomic studies in various biological samples.