Abstract: A method of HPLC for the determination of polyphenols in tobacco was proposed.The sample was extracted with methanol-H2O(80+20) solution by high-speed homogenization,and the extract obtained was defatted on MCI-GEL reversed phase resin SPE column.The polyphenols were separated by gradient elution with mixed solutions of methanol and acetic acid (1+99) of different ratios on Waters XTerra RP-18 column (3.9 mm×150 mm,5 μm),and determined at the wavelength of 345 nm with diode array detector (DAD).The main plant polyphenols in tobacco were successfully separated,giving values of recovery in the range of 95.2%-105.2% and RSD′s (n=7)in the range of 1.6%-3.0%.