Abstract: Ophthalmic solution of organic-inorganic layered double hydroxides hybrid nanocomposites based on layered double hydroxides(LDH)intercalated with pirenoxine sodium(PRN)and chitosan-glutathione(CG)was prepared, characterized and evaluated using rabbit precorneal retention. Mg-Al-PRN-LDH, Zn-Al-PRN-LDH and CG-PRN-LDH were synthesized by co-precipitation. The nanocomposites were characterized by laser particle sizer, powder X-ray diffraction(X-RD), fourier transform infrared spectra(FTIR)and transmission electron micrographs(TEM). The release of PRN from Mg-Al-PRN-LDH, Zn-Al-PRN-LDH, and CG-PRN-LDH nanocomposites and API in artificial tear was compared. Based on in vivo precorneal retention studies, PRN-LDH and CG-PRN-LDH nanocomposite dispersions showed significantly higher AUC(3. 72-, 7. 59-folds)and MRT(2. 18-, 2. 60-folds)than that of the commercial eye drops group. Organic-inorganic layered double hydroxides hybrid nanocomposites CG-PRN-LDH dispersions could remarkably improve precorneal retention of PRN.