Ryanodine receptor 2 (RyR2) is a Ca²⁺-release channel on the sarcoplasmic reticulum of cardiomyocyte that plays a central role in cardiac excitation-contraction coupling. Spontaneous Ca²⁺ release events caused by abnormal hyperactive RyR2 are linked to ventricular arrhythmias in patients with heart failure and catecholaminergic polymorphic ventricular tachycardia (CPVT). To search for novel RyR2 inhibitors from well-characterized drug library, we performed a high-throughput screening using HEK293 cells that stably express WT RyR2 and R-CEPIA1er, a genetically encoded endoplasmic reticulum (ER) Ca²⁺ indicator. By screening 1,535 compounds, we identified three ones that increased the ER Ca²⁺ signal greater than 4SD above the mean: chloroxylenol (Clxy), orserinate β-methyl (OBM) and riluzole (Ril). All three compounds increased ER Ca²⁺ signal and decreased the frequency of spontaneous Ca²⁺ oscillations dose-dependently in HEK293 cells expressing CPVT-linked mutant RyR2s as well as WT RyR2, which corresponded reduction of the Ca²⁺-dependent [³H]ryanodine binding. To investigate the drug effects on arrhythmogenic Ca²⁺ release, we observed Ca²⁺ signals in cardiomyocytes obtained from Tnnt2 ΔK210 and RYR2 I4093V mice that show Ca²⁺ waves, sparks and triggered activity frequently. Ril and Clxy suppressed Ca²⁺ waves without affecting action potential induced Ca²⁺ transients. These results suggest that the RyR2 inhibitors are promising candidates for novel anti-arrhythmic drugs.