D-Serine was detected in dialysate collected from the rat striatum using an online microdialysis-CE-LIF instrument. Dialysate can be analyzed every 12.5 s using the online instrument, giving much better temporal resolution than previously possible for D-serine. Basal concentrations of D-serine (8 ± 2 μM), glutamate (0.8 ± 0.2 μM), GABA (0.11 ± 0.04 μM), and L-serine (23 ± 4 μM) were measured. Increases in the concentrations of these neurochemicals induced by the introduction of high-K+ aCSF were quantitated. Notably, an increase in D-serine concentration in response to high-K+ aCSF was observed for the first time. The identity of the D-serine peak was confirmed unambiguously using D-amino acid oxidase to selectively remove D-serine from a dialysate sample. The microdialysis-CE-LIF instrument was able to monitor this enzymatic reaction as it proceeded over a period of 60 min, demonstrating that online microdialysis-CE-LIF is not only useful in monitoring in vivo dynamics but can also be used to monitor other chemical systems.