Proteins are composed exclusively of L-amino acids. Among elderly individuals, however, D-aspartic acid (D-Asp) residues have been found in eye lens and brain, as well as in other tissues. The presence of D-Asp may change the higher-order structure of a protein, which in turn may have a role in age-related disorders such as cataract and Alzheimer's disease. D-Asp results from the spontaneous racemization of Asp residues in susceptible proteins. During aging, natural Lα-Asp residues in proteins are non-enzymatically isomerized via a succinimidyl intermediate to L-β-, D-α- and D-β-isomers. This isomerization does not happen uniformly, but instead occurs at specific residues that are susceptible to isomerization due to their sequence or structural context. Thus, it is necessary to establish the nature of each individual Asp residue in susceptible proteins. Recently, a new method based on LC-MS/MS for the analysis of Asp isomerization at specific protein sites has been described. In this review, we first show that the homochirality of amino acids in proteins is not guaranteed throughout life. We then describe the development of a new method for protein-bound D-amino acid analysis, and discuss the negative influence that D-Asp has on protein structure and function.