The aim of the present work was to investigate if chitosan salification with ascorbic acid could produce an increase in chitosan penetration enhancement properties towards buccal mucosa and intestinal Caco-2 cell monolayer. Three different chitosan grades were considered. Chitosan hydrochloride and lactate were used as references. Fluorescein isothiocyanate-dextran (FD4), a hydrophilic high MW molecule, was employed as model penetrant. Chitosan ascorbate showed better penetration enhancement properties towards both buccal porcine mucosa and Caco-2 cell monolayer with respect to hydrochloride and lactate salts. Cytotoxicity of chitosan ascorbate assessed on Caco-2 cells was comparable with those of chitosan hydrochloride and lactate.