Chiral amino acid analysis is currently gathering attention since several d-form amino acids (minor enantiomers of the predominantly observed l-amino acids) have been demonstrated to be the new physiologically active substances and biomarkers. However, the amounts of these d-amino acids in biological samples are trace in most cases, and sensitive and selective analytical methods are essential for their determination. In the present chapter, multidimensional liquid chromatography (LC) systems, especially the design and development of two-dimensional LC–tandem mass spectrometry (MS/MS) systems, are summarized, and the applications of the methods to the determination of chiral amino acids in various real-world matrices, including human urine, plasma, fermented products, and protein samples, are described.