D: -Amino acids play several key roles and are widely diffused in living organisms, from bacteria (in which D-alanine is a component of the cell wall) to mammals (where D-serine is involved in glutamatergic neurotransmission in the central nervous system). The study of the biological processes involving D-amino acids and their use as clinical or biotechnological biomarkers requires reliable methods of quantifying them. Although "traditional" analytical techniques have been (and still are) employed for such tasks, enzymatic assays based on enzymes which possess a strict stereospecificity (i.e., that are only active on the D-enantiomers of amino acids) allowed the set-up of low-cost protocols with a high sensitivity and selectivity and suitable for determining the D-amino acid content of complex biological samples. The most exploited enzyme in these assays is D-amino acid oxidase, a flavoenzyme that exclusively oxidizes D-amino acids and possesses with a broad substrate specificity and a high kinetic efficiency.