BACKGROUND: – Mutations in the KCNJ5 (potassium inwardly rectifying channel subfamily J member 5) gene, encoding an inwardly rectifying potassium channel, can drive aldosterone overproduction in a subset of aldosterone-producing adenomas and in familial hyperaldosteronism type III. Our objective was to identify small molecule compounds that specifically antagonize mutant KCNJ5 channels. METHODS: – Virtual screening of over 6 million small molecules identified compounds that putatively bind to KCNJ5 channels. The effect of 108 of these candidates was evaluated in vitro in human adrenocortical cells (HAC15) with inducible expression of wild-type or mutated forms of KCNJ5. Assessment encompassed cell viability, flow cytometry, gene expression, and adrenal steroid quantification via liquid chromatography–tandem mass spectrometry. RESULTS: – Compounds antagonizing mutated KCNJ5 function were identified by evaluating their ability to rescue adrenal cell death induced by overexpression of mutant KCNJ5. A spiroquinoline compound, referred to as compound 81 (C81), effectively rescued cell death induced by KCNJ5 L168R in both monolayer and spheroid HAC15 cell cultures. C81 treatment caused a 69% to 85% reduction in CYP11B2 (aldosterone synthase) mRNA levels induced by KCNJ5 L168R, G151R, or T158A expression, compared with untreated cells. C81 also reduced aldosterone secretion by 65% in cells expressing KCNJ5 L168R and decreased 18-oxocortisol and 18-hydroxycortisol production by 78% and 90%, respectively. However, C81 had no significant effect on steroid secretion in cells overexpressing wild-type KCNJ5. CONCLUSIONS: – C81 shows potential as a small molecule antagonist to specifically target pathological aldosterone secretion in familial hyperaldosteronism type III or KCNJ5-mutated aldosterone-producing adenomas. These findings suggest new avenues for genotype-based primary aldosteronism diagnostics and targeted treatments, contributing to personalized patient care.