In order to comprehensively screen and identify the glycoside compounds in tobacco, a simple, rapid and sensitive method of high performance liquid chromatography/electrospray ionization linear ion-trap tandem mass spectrometry (HPLC-ESI-LIT/MS n ) coupled with electrospray ionization orbitrap mass spectrometry (ESI-Orbitrap-MS) was developed for the first time. As a result, twenty-two glycoside compounds, including eleven alcoholic glycosides, eight phenolic glycosides, two ester glycosides and an indole glycoside, were reliably identified from tobacco with high mass accuracy (within 5 mDa). Among them, four compounds were confirmed as novel molecules and other four compounds, as far as we know, were not reported previously in tobacco. This study provided a useful tool to identify the new structures of glycoside compounds in natural products, especially when there were no reference compounds available.