The NMDA subtypes of glutamatergic receptors (NMDARs) are unusual in that their activation requires the binding of both glutamate and a co-agonist glycine or d -serine. Whereas glycine was first suggested to play such a role, it was later established that d -serine could serve as an endogenous co-agonist at different central synapses. We still do not know the exact nature of the endogenous co-agonist(s) of NMDARs and the function of the so-called glycine B site in many brain structures. We introduced few years ago the use of enzymes that specifically degrade either d -serine or glycine to decipher the influence of these amino acids on NMDA receptors function. The use of these enzymatic scavengers represents an invaluable technique for neurophysiologists investigating the neuromodulation of the glycine B site in the CNS. Here, we describe the proper way to manipulate these enzymes during electrophysiological recordings in acute brain slices and highlight the experimental tricks.