Proper water management is required for the operation of polymer electrolyte fuel cells (PEFCs), in order to maintain the critical balance between adequate membrane hydration and prevention of water flooding in the catalyst layer. In PEFCs, the membrane electrode assembly (MEA) is sandwiched between two gas diffusion layers (GDLs). In addition, a microporous layer (MPL) is generally applied to the GDL substrates for better water removal from the cathode catalyst layer. This paper is the first to report on an ex situ characterization method for water flooding in GDLs. As the humidity of O2 gas on the substrate side of the GDL was increased in incremental steps, O2 gas began to diffuse into the MPL side of the GDL. When the O2 relative humidity exceeded the dew point, water flooding was observed on the surface of the MPL and the O2 concentration dropped sharply because the O2 diffusion was suppressed by the produced liquid water. When comparing to the estimated mass transfer loss based on the actual polarization curves of an MEA using the GDL, it was found that the decrease in the O2 concentration on the MPL side of the GDL can be used as an index of water flooding in the PEFC.