Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting alpha-subunits with specific regulatory beta-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of alpha- and beta-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K(+) channel inhibitor and its iterative application to tally the number of beta-subunits in a KCNQ1/KCNE1 K(+) channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K(+) channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE beta-subunit modulation of voltage-gated K(+) channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K(+) channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE beta-subunits.