Abstract: The effect of 3,5-dimethylphenyl isocyanate on the chiral recognition of hydroxypropyl-β-cyclodextrin bonded stationary phase has been studied. Hydroxypropyl-β-cyclodextrin, bonded to 3-aminopropyltriethoxysilane through (3-isocyanatopropyl) triethoxysilane was used as the coupling agent, then other hydroxyl groups of β-CD and silica gel were derivatized with 3,5-dimethylphenyl isocyanate, thus a 3,5-dimethylphenylcarbamate perderivatized hydroxypropyl-β-cyclodextrin bonded silica gel stationary phase was prepared. The chiral resolution of nine chiral drugs in reversed phase chromatography showed that 3,5-dimethylphenylcarbamate perderivatized hydroxypropyl-β-cyclodextrin bonded stationary phase has better separation result than that of hydroxypropyl-β-cyclodextrin bonded stationary phase.