D-amino acids, the enantiomers of L-amino acids, are candidates to be novel physiologically active substances and the biomarkers in mammals. However, the amounts of D-amino acids in the tissues and physiological fluids are extremely small in most cases, and sensitive and selective analytical methods are needed for their determination. In the present manuscript, we review the analytical technologies including our recent advances for the determinations of small amounts of D-amino acids in mammals and the applications to clarify their physiological meanings.