D-Serine is localized in the mammalian forebrain and modulates brain functions as a coagonist of an N-methyl-D-aspartate receptor. D-Serine is also found in human urine, although its physiological meaning is unclear. A method for rapid and simple assay of D-serine is probably useful for studying its physiological role and clinical relevance. Currently, D-serine is assayed by high-performance liquid chromatography after derivatization of the amino acid to a diastereomer. The method is time consuming and requires expensive equipment. In this study, we developed a rapid and simple method for the D-serine assay using D-serine dehydratase newly found in Saccharomyces cerevisiae. The yeast d-serine dehydratase acts dominantly on d-serine, in contrast with previously reported bacterial enzymes that act on both D- and L-serine. In our method, pyruvate produced from D-serine by the dehydratase reaction is assayed with lactic dehydrogenase and reduced nicotinamide adenine dinucleotide or with 2,4-dinitrophenylhydrazine. Our enzymatic method could be used for the quantitative determination of D-serine in human urine.