Multidimensional separation systems offer several advantages over traditional one-dimensional separation systems, particularly their ability to separate molecules from complex mixtures. Two-dimensional liquid chromatography (2D-LC) significantly enhances the ability to analyze complex mixtures by providing greater separation power, sensitivity, and flexibility, making it an invaluable tool for metabolomics research. The 2D-LC is an exciting mode when pursuing untargeted analysis, as it allows for high-resolution separation and subsequent identification and quantification of more analytes. This chapter summarizes the current applications of 2D-LC in metabolomics and the setups of different separation modes that are being employed, presenting the most suitable combinations of chromatographic methods for different targeted and untargeted metabolomics applications.