A microcolumn high performance liquid chromatography (HPLC) and mass spectrometry method for the determination of polyphenols in tobacco is studied. The polyphenols can be extracted from tobacco by refluxing in a boiling water bath with 80% methanol and degreased by solid phase extraction with a C18 cartridge. Chlorogenic acid, rutin, scopoletin, caffeic acid, scopolin, esculetin, quercetin, kaempferol-3-rutinoside and other polyphenols are satisfactorily separated on a Waters XterraTM RP18 (1.0 x 50 mm, 2.5 µm) microcolumn. A gradient elution is used with methanol and 1% acetic acid solution at a flow-rate of 0.1 ml/min with detection by photodiode array between 200-400 nm. The composition of the mobile phase was: A (methanol) and B (1% acetic acid) for 0 min (20%A:80%B), 3 min (80%A:20%B), 4 min (80%A:20%B) in a linear ramp. The limits of detection are: 52 ng/ml for chlorogenic acid, 18 ng/ml for caffeic acid, 34 ng/ml for esculetin, 32 ng/ml for scopolin, 27 ng/ml for scopoletin, 56 ng/ml for rutin and 48 ng/ml for kaempferol-3-rutinoside. The key polyphenols in tobacco are identified by comparing the retention time, the UV-spectra, and the mass spectra with those of the standard isomers of chlorogenic acid. The recovery of tobacco polyphenols is 95~104% and the relative standard deviations are 1.4~1.9%. This method has been successfully applied to qualitatively and quantitatively analyse polyphenols in tobacco with good results.