The durability of the polymer electrolyte membrane fuel cell is identified as one of the major limiting factors for the wide commercialization of fuel cells. In particular, the phenomenon of Ru crossover from the anode to the cathode has severe performance implications. In the present investigation, a facile and cost-effective in situ diagnostic method was developed to quantify Ru crossover and to predict its performance impact. The scheme was validated with neutron activation analysis (NAA). A proprietary anode accelerated stress test was used to cause various degrees of Ru crossover, and the end-of-life cathode catalyst layers were characterized by cyclic voltammetry, X-ray diffraction, and NAA. The observed characterization relationships provided a mechanistic understanding of Ru crossover and were demonstrated to be functional as an in situ diagnostic method.