Most of physiologically active substances are chiral, and their enantiomers exhibit different biological properties. Separation of such chiral molecules by ion mobility spectrometry (IMS) enables rapid analysis or detailed distribution analysis using mass spectrometry. In addition, recent progress in the resolution of IMS enables separation of various chiral molecules from their enantiomers. Here, we briefly introduce the methodology and our recent method to discriminate a target chiral molecule from its enantiomer by IMS.