Metabolomics has been an evolving science with a wide range of applications in various fields. However, previous studies have rarely focused on metabolite chirality. In this study, to achieve metabolic profiling of chiral amino acids and related metabolites, we developed a high-throughput method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The combination of two types of chiral columns (with binaphthyl-based crown ether and cinchona alkaloid-derived zwitterionic stationary phases) enabled the analysis of 115 chiral and non-chiral metabolites. By finely optimizing MS/MS parameters, the method allowed the highly sensitive (0.001-50 nmol/mL) and wide dynamic range detection of targeted analytes in a standard solution without derivatization. We applied the method to food samples (cheese), and successfully quantified trace levels of metabolites such as n-amino acids in samples. Additionally, we performed principal component analysis on the metabolome data and obtained unique profiles that reflected metabolite chirality. These results demonstrated the applicability and feasibility of the LC-MS/MS method as an effective tool for wide-targeted chiral metabolome analysis. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.