The association between the kainate receptors (KARs) GluK1 and GluK2 and the modifying proteins neuropilin- and tolloid-like 1 (NETO1), neuropilin- and tolloid-like 2 (NETO2), and postsynaptic density protein 95 (PSD95) is likely to produce distinct GluK1 and GluK2 pharmacology in postsynaptic neurons. However, little is known about their corresponding modulatory effects on GluK1 and GluK2 activity in high-throughput assays for cell-based drug discovery. Using heterologous cells that potentially mimic the response in native cells in a fluorescence imaging plate reader (FLIPR) assay, we have investigated assays that incorporate (1) coexpression of GluK1 or GluK2 with their modulatory proteins (NETO1, NETO2, PSD95) and/or (2) enablement of assays with physiological concentration of native GluK1 and GluK2 agonist (glutamate) in the absence of an artificial potentiator (e.g., concanavalin A [Con A]). We found that in the absence of Con A, both NETO1 and NETO2 accessory proteins are able to potentiate kainate- and glutamate-evoked GluK1-mediated Ca(2+) influx. We also noted the striking ability of PSD95 to enhance glutamate-stimulated potentiation effects of NETO2 on GluK1 without the need for Con A and with a robust signal that could be utilized for high-throughput FLIPR assays. These experiments demonstrate the utility of heterologous cells coexpressing PSD95/NETO2 with GluK1 or GluK2 in native cell-mimicking heterologous cell systems for high-throughput assays and represent new avenues into the discovery of KAR modulating therapies.