This work presents a new concept for realising a reference electrode configuration in a PEM fuel cell by means of laser ablation. The laser beam is used to evaporate a small part of the electrode of a catalyst-coated membrane (CCM) to isolate the reference electrode from the active catalyst layer. This method enables the simultaneous ablation of the electrodes on both sides of the CCM because the membrane is transparent for the laser beam. Therefore, a smooth electrode edge without electrode misalignment can be realised. A test fuel cell was constructed which together with the ablated CCM enables the separation of the total cell losses during operation into the cathode, anode and membrane overpotentials in PEFC as well as in DMFC mode. The methanol tolerance of a selenium-modified ruthenium-based catalyst (RuSe (x) ) was investigated under real fuel cell conditions by measuring polarisation curves, electrochemical impedance spectroscopy (EIS) and current interrupt measurements (CI).