D-Serine is a transmitter-like molecule that physiologically activates NMDA receptors in the brain. Although D-serine was thought to be exclusively released by astrocytes, we recently demonstrated endogenous D-serine release from neurons in cultures and slices. So far high-performance liquid chromatography (HPLC) has been the standard technique to monitor D-serine and other amino acids. This method employs pre-column derivatization with a chiral reagent to produce fluorescence derivatives that can be further separated on a reversed-phase column. Due to the close retention times of L-serine, L-glutamine, and D-serine, the quantification of low levels of endogenous D-serine synthesis and release from cell cultures and tissues can be challenging. We here describe an enzymatic treatment method to specifically destroy L-glutamine and L-serine by glutaminase and L-serine dehydratase enzymes, respectively, allowing accurate determination of nanomolar D: -serine concentrations by subsequent HPLC analysis.