d-Amino acids are currently recognized as naturally occurring physiologically active substances and biomarkers in mammals. The progress of analytical technologies, mostly high resolution chromatographic or electrodriven separation methods, has significantly contributed to the advances in d-amino acid research in real biological matrices. In this review, we would like to describe the d-amino acid research, from the discovery of appreciable amounts of free d-amino acids in mammals to the current metabolomics study focusing on amino acid enantiomers. The liquid phase enantioselective analytical methods utilized for the determination of d-amino acids in mammals including human beings will be discussed.