N-Methyl-D-aspartate receptors (NMDAR), a class of ionotropic glutamate receptors, play a critical role in synaptic transmission and plasticity in the central nervous system. A unique subtype of these receptors, GluN1/3A, predominantly expressed during early brain development, has been recently proposed as an excitatory glycine receptor. Our investigation aims to deliver a comprehensive characterization of the mechanism of action of recently reported selective GluN3A inhibitors by using a stable cell line expressing recombinant GluN1/3A receptors. Furthermore, we are describing an assay for evaluating the activity of these inhibitors at native receptors in cultured primary neurons. For measuring functional GluN3A responses we used Ca2+ imaging performed at single-cell or high-throughput format. For looking into the mechanism of action of GluN3A inhibitors, we performed curve-shift experiments of either CGP-78608, a GluN1 specific inhibitor, or Glycine, the endogenous GluN3A agonist, in the presence of the test compounds. Selective GluN1 antagonists (DCKA, 7-CKA) demonstrated a competitive mode of action at the GluN1 subunit while the GluN3A inhibitors (EU1180-E438, WZB117) showed a non-competitive profile.In primary neurons, we identified a specific neuronal population that displayed functional GluN3A responses, distinguishable from Glycine receptor or classical NMDAR responses, which could be blocked by selective inhibitors. The potency data measured by Ca2+ flux recordings in primary neurons aligned well with the IC50 values determined in the cell line expressing recombinant GluN1/3A.Our study provides deeper insights into the pharmacology of known GluN1/3A modulators and the validation of a novel neuronal assay for confirming activity of compounds at native receptors.