Glucocorticoids (GCs)-ligands of the glucocorticoid receptor (GR)-are widely used to treat inflammatory diseases, but suffer from significant side effects and poor responsiveness in certain patient populations. Identification of chemical GR modulators may provide insights into the regulatory mechanisms of anti-inflammatory functions of GR and help improve GC-based therapy. Here we report the development and application of a high-throughput screening to identify compounds that either enhance or suppress the anti-inflammatory effect of GR function. Using a cell-based GR activity assay that measures Dexamethasone (Dex)-mediated NF-κB repression, we have screened ~8,000 compounds and identified several compounds that suppressed GR activity, including multiple GSK3β inhibitors and anti-cancer agent camptothecin. Notably, we also identified two kinase IKK2 inhibitors, including TPCA-1, as GR enhancers that improve the anti-inflammatory effect of GR. In particular, TPCA-1 augmented the activity of Dex in NF-κB repression by attenuating GR down-regulation. Consistent with the observation, siRNA-mediated IKK2 knockdown decreased GR down-regulation and increased GR expression. Together, our results identified chemical compounds as novel modulators of GR and revealed an unexpected role for IKK2 in GR down-regulation. Furthermore, we have established a high-throughput screening platform for discovering GR-modulating compounds that may be repurposed to improve current GC-based therapies.