Recently, the demand for D-amino acid profiling has been drastically increasing because the significance of D-amino acid in various biological events is suggested. However, the present methodologies for D-amino acid profiling are still unsatisfactory. Therefore, a highly sensitive, robust, high-throughput, and user-friendly method for D-amino acid profiling must be developed. In this paper, we developed a novel method for D-amino acid profiling using a combination of a chiral column and time of flight mass spectrometry (TOMS). To our knowledge, our approach has the best performance for 0-amino acid analysis that includes the shortest analytical time (within 10 min), the highest enantioseparability without derivatization, and the largest coverage for analytical targets (more than one hundred targets including non-proteinogenic amino acids and amines). Thus, our novel profiling method will be instrumental in advancing the D-amino acid research in the future. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.