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Abstract Bioelectronics based on organic mixed conductors offers tremendous application potential in biological interfacing, drug delivery systems, and neuromorphic devices. The ion injection and water swelling upon electrochemical switching can significantly change the molecular packing of polymeric mixed conductors and thus influence the device performance. Herein, we quantify ion and water injection, and analyze the change of microscopic molecular packing of typical polymeric mixed conducting materials, namely poly(3,4‑ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) and poly(2‐(3,3′‐bis(2‐(2‐(2‐methoxyethoxy)ethoxy) ethoxy)‐[2,2′‐bithiophen]‐5‐yl)thieno[3,2‐b]thiophene) (p(g2T‐TT)), by integrating electrochemical quartz crystal microbalance with dissipation monitoring, in situ charge accumulation spectroscopy, and electrical current‐voltage measurement. The penetration of ions and water can lead to viscous and disordered microstructures in organic mixed conductors and the water uptake property plays a more dominant role in morphological disruption compared with ion uptake is demonstrated. This study demonstrates the potential application of the combined optical, gravimetric, and electrical operando platform in evaluating the structural kinetics of organic mixed conductors and highlights the importance of concertedly tuning the hydration process, structural integrity, and charge transport properties of organic mixed conductors in order to achieve high performance and stable bioelectronic devices.