Active species crossover continues to frustrate durational performance for redox flow batteries (RFBs), requiring thorough evaluation of membrane / separator properties. Characterization workflows typically employ a suite of ex situ experimental techniques, but these approaches do not capture the dynamic conditions (e.g., variable concentrations, alternating polarity) encountered in redox flow cells. Here, we report a facile method for assessing crossover directly in redox flow cells—compositionally unbalanced symmetric cell cycling (CUSCC). Based on conventional symmetric cell cycling, CUSCC imposes a concentration gradient between two chemically similar half-cells, inducing species crossover during galvanostatic cycling, which results in a characteristic “capacity gain” over time. We first develop a zero-dimensional model to describe fundamental processes that underpin the technique and examine the dependence of capacity gain on membrane / separator properties and operating conditions. Subsequently, we perform proof-of-principle experiments using FeCl2 / FeCl3 and Nafion 117 as a representative system and demonstrate results consistent with those predicted from simulations. Finally, we use model fits of the capacity gain data to extract membrane transport parameters, obtaining similar values to those measured from ex situ techniques. Overall, this work describes a promising new approach for characterizing species crossover and expands the RFB testing toolbox.