As shown in recent work, thin layer ion-selective multi-ionophore membranes can be interrogated by cyclic voltammetry to detect the ion activity of multiple species simultaneously and selectively. Additional fundamental evidence is put forward on ion discrimination with thin multi-ionophore-based membranes with thicknesses of 200 ± 25 nm and backside contacted with poly-3-octylthiophene (POT). An anodic potential scan partially oxidizes the POT film (to POT+), thereby initiating the release of hydrophilic cations from the membrane phase to the sample solution at a characteristic potential. Varying concentration of added cation-exhanger demonstrates that it limits the ion transfer charge and not the deposited POT film. Voltammograms with multiple peaks are observed with each associated with the transfer of one type of ion (lithium, potassium, and sodium). Experimental conditions (thickness and composition of the membrane and concentration of the sample) are chosen that allow one to describe the system by a thermodynamic rather than kinetic model. As a consequence, apparent stability constants for sodium, potassium, and lithium (assuming 1:1 stoichiometry) with their respective ionophores are calculated and agree well with the values obtained by the potentiometric sandwich membrane technique. As an analytical application, a membrane containing three ionophores was used to determine lithium, sodium, and potassium in artificial samples at the same location and within a single voltammetric scan. Lithium and potassium were also determined in undiluted human plasma in the therapeutic concentration range.