Asymmetric separators with polysulfide barrier properties, consisting of porous polypropylene (PP) grafted with styrene sulfonate (SS), PP-g-PLiSS, were characterized in symmetric Li/Li cells using electrochemical impedance spectroscopy to investigate the influence of separator chemistry on the Li electrode/electrolyte interface. The symmetric Li/Li cell approach was found to be applicable for probing the transport properties of the separator and, therefore, determining the role of the functionality added to the separator. Electrolyte resistance and the formation of a surface film on metallic Li were both monitored as functions of time and the concentration of cation-exchange groups introduced at and near the surface of one side of the separator (expressed as the graft level). No continuous build-up of Li electrode/electrolyte interface resistance was observed, indicating that the addition of a cation-exchange layer did not hinder entirely Li-ion transport through the separator. In addition, the microstructures of the separators were reconstructed based on focused ion beam/scanning electron microscopy tomography to determine the effective ionic conductivity and effective tortuosity of the PP-g-PLiSS separators. These values showed that ion mobility within the separator changed with increasing graft level, indicating that the concentration of SS groups and bulk porosity of the separator have to be adjusted to maintain the effective conductivity at a practical level.