While organic mixed ionic/electronic conductors are widely studied for various applications in bioelectronics, energy generation/storage, and neuromorphic computing, a fundamental understanding of the interactions between the ionic and electronic carriers remains unclear, particularly in the wet state and on electrochemical cycling. Here, we show that operando NMR spectroscopy can selectively probe and quantify ion and water movement during the doping/dedoping of poly(3,4-ethylene dioxythiophene) poly(styrene sulfonate) (PEDOT:PSS) films, the most widely used organic mixed conductor. Na+ ions near or within the PEDOT-rich domains experience an anisotropic environment resulting from the underlying partial PSS chain orientation in the polymer films, giving rise to a distinct quadrupolar splitting in the 23Na NMR spectrum. Operando 23Na NMR studies reveal a linear correlation between the quadrupolar splitting and the charge stored in the film, which is interpreted in terms of the roles that the Na+ ions at the PEDOT/PSS interfaces play in charge balance and electric double layer formation. The observed correlation is quantitatively explained by a competitive binding model, in which holes on the PEDOT backbone are bound to PSS, the hole concentration changes during doping/dedoping inducing variations in the Na+ binding percentage at the PEDOT/PSS interfaces. The Na+-to-electron coupling efficiency, measured via 23Na NMR intensity changes, varies noticeably depending on the cycling history of the film. Operando 1H NMR spectroscopy confirms that water molecules accompany the ions that are injected into/extracted from the films. These findings shed light on the working principles of organic mixed conductors and demonstrate the utility of operando NMR spectroscopy in revealing structure-property relationships in electroactive polymers.