AbstractChiral analysis is of high interest in many fields such as chemistry, pharmaceuticals and metabolomics. Mass spectrometry and ion mobility spectrometry are useful analytical tools, although they cannot be used as stand-alone methods. Here, we propose an efficient strategy for the enantiomer characterization of amino acids (AAs) using non-covalent copper complexes. A single ion mobility monitoring (SIM²) method was applied on a TIMS-ToF mass spectrometer to maximize the detection and mobility separation of isomers. Almost all of the 19 pairs of proteinogenic AA enantiomers could be separated with at least one combination with the chiral references L-Phe and L-Pro. Furthermore, we extended the targeted SIM² method by stitching several mobility ranges, in order to be able to analyze complex mixtures in a single acquisition while maintaining high mobility resolution. Most of the enantiomeric pairs of AAs separated with the SIM² method were also detected with this approach. The SIM² stitching method thus opens the way to a more comprehensive chiral analysis with TIMS-ToF instruments. Graphical Abstract