Occurrence of d-amino acids in living organisms is a useful indicator of various changes, diseases, or disorders. Determination of amino acid enantiomers, namely the enantiomeric ratio of amino acids or excess of certain d-amino acids, represents a useful tool in the studies of aging processes or biomarkers in disease/disorder diagnosis in humans. The amount of d-amino acids is usually very low. Therefore, suitable sample pretreatment, often derivatization, and highly selective and sensitive separation methods are essential for d-amino acid analysis in this field. Chromatographic techniques offer appropriate choices for solving these tasks. This review covers the advances in methodology and development of improved instrumental chromatographic methods focused on d,l-amino acid separation and determination. New findings in the area of possible d-amino acid biomarkers are also included.