Through-plane potential and overpotential distributions in a vanadium redox flow battery (VRFB) is investigated in-situ using saturated Ag/AgCl reference electrodes attached to each side of the membrane with edge-type configuration. Combined with electrochemical impedance spectroscopy (EIS), this operando approach enables detailed quantification of the voltage losses contributed from the individual VRFB cell components with the magnitudes of ohmic and charge transfer losses at practical charge-discharge conditions. It is found that the overpotentials for vanadium redox reactions on graphite felt electrodes at the positive and negative half-cells are one order magnitude larger than the voltage loss through the typical cation-exchange membrane (Nafion®117) for charging-discharging at current densities from 50 to 120 mA cm−2. Within the overall half-cell overpotentials, the ohmic voltage loss accounts for about 40%. The analysis reveals that the voltage loss by the anode charge transfer reaction is about three folds larger than that by the cathode reaction, suggesting that sluggish V2+/V3+ redox kinetics in the negative half-cell is the major factor limiting the overall VRFB cell performance. This operando methodology is effective for the in-depth analysis of VRFB performance in practical conditions as well as the development of high-performance cell components and efficient total cell design by a collective and quantitative approach.