Abstract: A method for glycosides detection in tobacco leaves by heart-cutting Rp/Rp two-dimensional liquid chromatography-tandem mass spectrometry was established. Three glycosides were determined, including 3-oxo-7,8-dihydro-α-ionol-β-D-glucopyranoside, 3-oxo-α-ionol-β-D-glucopyranoside and 3-hydroxy-β-dihydro-damascone-β-D-glucopyranoside. Only extraction and filtration were required in sample pretreatment. The RSDs of intra-day and inter-day were in the range of 1.0%-3.2% and 2.8%-8.4%, respectively, which showed good reproducibility. Since sample pretreatment was very simple, the method was quite suitable for daily detection. Different types of tobacco were determined, including Virginian-type tobacco, Burley tobacco, oriental tobacco and sun-cured tobacco. The results revealed that glycosides in flue-cured and fresh Virginian-type tobacco were significantly higher than other types, which agreed well with previous reported articles. 157 kinds of Virginian-type tobacco samples from different regions were monitored and investigated. Among the factors, which influenced contents of glucosides in Virginian-type tobacco, sugar was no longer the major one, catabolite of carotenoids (C13-isoprenol glucosides) and temperature were also considered an important factor in our study.