Neuronally triggered phosphorylation monotonically and cyclably drives the assembly of reflectin proteins, resulting in the fine-tuning of colors reflected from specialized skin cells in squid for camouflage and communication. Reflectin has been found as the primary constituent within stacks of plasma membrane-bounded platelets (iridosomes) found in iridocyte cells. This cellular ‘superstructure’ consisting of alternating layers of high refractive index platelets and low refractive index extracellular space effectively functions as a biological Bragg reflector. In vivo, reflectin assembly triggers osmotic dehydration of the Bragg lamellae, reducing the platelet thickness, and thus dynamically tuning the color of the reflected light. Our previous study has shown that electrochemistry, used as in vitro surrogate for in vivo phosphorylation-mediated charge neutralization, triggers voltage-calibrated and cyclable control of the rate and size of reflectin assembly. This process is highly similar to the observed physiological behavior. Here, we demonstrate for the first time that electrochemical reduction enables tunable and reversible control of refractive index and thickness of a reflectin thin film formed by drop-casting on a conductive substrate to mimic the platelet structures in cephalopod iridophores. Electrochemically driven in situ spectroscopic ellipsometry was developed to trigger and simultaneously analyze the change in optical properties of the reflectin film and thus further elucidate the color-changing mechanisms in squid skin. Our results confirm the hypothesis that the extent of charge neutralization controlled by applied voltage plays a role in calibrating the water volume fraction within platelets and the resulting optical features. This work opens new means for analyzing the biophysical mechanisms regulating color change by reflectin assembly, designing advanced functional materials and devices, and bridging the biotic–abiotic interface.