Homochirality is essential for life. For a long time, it was considered that d-amino acids were excluded from living systems. In the past 30 years, however, d-amino acids have been found in living organisms in the form of free amino acids, peptides and proteins, owing to advances in the analysis of optical isomers of amino acids. Free d-amino acids and d-amino-acid-containing peptides have been shown to have important physiological functions. The amount of d-aspartate (Asp) residues in protein spontaneously increases in metabolically inert tissues such as the eye and brain during aging, and may be related to cataract formation and the development of Alzheimer disease, suggesting that d-Asp might be a molecular marker of aging and age-related disorders. The presence of d-Asp in living organisms is thought to result from the isomerization of l-Asp residues in some proteins. Furthermore, the isomerization of Asp does not occur uniformly but only at specific sites. Therefore, it is necessary to determine the sites of isomeric Asp in these proteins in order to elucidate the mechanism of spontaneous Asp isomerization during aging. Herein, we summarize the localization and mechanism of d-amino acids in proteins of living tissues, and the effects of d-amino acid formation in proteins. Furthermore, we describe methods for the analysis of protein-bound d-amino acids including a conventional enantioseparation method based on HPLC and a new convenient method based on LC–MS that can identify the specific sites of d-Asp in proteins.