A reliable and rapid method based on high-performance liquid chromatography (HPLC-UV) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) has been developed for the characterization and quantification of solanesol in extracts of tobacco leaves from different sources. The solanesol was extracted from tobacco leaf via saponification and ultrasonic-assist extraction, and the extraction conditions were optimized. The HPLC conditions are as following: Hypersil C4 (4.6 mm × 150 mm, 5 μm) column, acetonitrile and water as mobile phase, flow-rate is 0.8 ml/min, detection length of UV is 202 nm, injection volume is 10 μl. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of solanesol in tobacco leaves with a linear dynamic range of 3.65–4672 ng, a detection limit of 1.83 ng, and an average recovery of 98.7%. The method has been applied to analyze and compare different tobacco samples. The results show that the solanesol content in samples of different geographic locations varies widely from 0.20 to 1.50%. When different parts of the tobacco plant are compared, the top parts of the leaves are more abundant in solanesol content than those of lower parts.