Cell-penetrating peptides (CPPs) are short (<30 amino acids), generally cationic, peptides that deliver diverse cargos into cells. CPPs access the cytosol either by direct translocation through the plasma membrane or via endocytosis followed by endosomal escape. Both direct translocation and endosomal escape can occur simultaneously, making it non-trivial to specifically study endosomal escape alone. Here we depolarize the plasma membrane and showed that it inhibits the direct translocation of several CPPs but does not affect their uptake into endosomes. Despite good endocytic uptake many CPPs previously considered to access the cytosol via endosomal escape, failed to access the cytosol once direct translocation was abrogated. Even CPPs designed for enhanced endosomal escape actually showed negligible endosomal escape into the cytosol. Our data reveal that cytosolic localization of CPPs occurs mainly by direct translocation across the plasma membrane. Cell depolarization represents a simple manipulation to stringently test the endosomal escape capacity of CPPs.