D-serine is the major D-amino acid in the mammalian central nervous system. As the dominant co-agonist of the endogenous synaptic NMDA receptor, D-serine plays a role in synaptic plasticity, learning, and memory. Alterations in D-serine are linked to neuropsychiatric disorders including schizophrenia. Thus, it is of increasing interest to monitor the concentration of D-serine in vivo as a relevant player in dynamic neuron-glia network activity. Here we present a procedure for amperometric detection of D-serine with self-referencing ceramic-based microelectrode arrays (MEAs) coated with D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We demonstrate in vitro D-serine recordings with a mean sensitivity of 8.61 +/- 0.83 pA/mu M to D-serine, a limit of detection (LOD) of 0.17 +/- 0.01 mu M, and a selectivity ratio of 80:1 or greater for D-serine over ascorbic acid (mean +/- SEM; n = 12) that can be used for freely moving studies.