In this study, a membrane electrode assembly(MEA) composed of three electrodes(anode, cathode, and reference electrode) is designed to investigate the effects of methanol concentration on the overpotentials of anode and cathode in direct methanol fuel cells(DMFCs). Using the three-electrode cell, in-situ analyses of the overpotentials are carried out during direct methanol fuel cell operation. It is demonstrated that the three-electrode cell can work effectively in transient state operating condition as well as in steady-state condition, and the anode and cathode exhibit different overpotential curves depending on the concentration of methanol used as fuel. Therefore, from the real-time separation of the anode and cathode overpotentials, it is possible to more clearly prove the methanol crossover effect, and it is expected that in-situ analysis using the three-electrode cell will provide an opportunity to obtain more diverse results in the area of fuel cell research.