Kir2.1 has been implicated in a number of channelopathies including Andersen-Tawil Syndrome, Short QT Syndrome and catecholaminergic polymorphic ventricular tachycardia. We developed a mass spectrometry-based assay to identify phosphorylation sites and test the functional consequence of removing those sites by site-directed mutagenesis. Our study identified novel sites of phosphorylation and suggests that the site of phosphorylation can influence channel function. We envision our approach can be readily adapted to study additional mutations and other ion channels.