A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) was developed for the determination of solanesol in Nicotiana tabacum. Sample preparation was performed by ultrasonic extraction with methanol for 20 min and then supernatant was extracted with hexane. The method used atmospheric pressure chemical ionization (APCI) detection in positive-ion mode. The separation of solanesol was performed on a Symmetry Shield RP18 column with a mixture of acetonitrile and isopropanol (1:1, v/v) containing 2mM ammonium acetate as mobile phase. Quantification of solanesol was performed by the standard addition method. The limit of quantification (LOQ) and limit of detection (LOD) of solanesol were, respectively, 5.0 ng/ml (S/N=10) and 1.5 ng/ml (S/N=3). The relative standard deviations of peak area were 0.89 and 1.12% for intra-day and inter-day, respectively. The recoveries of solanesol ranged from 97.72 to 99.67% and the corresponding R.S.D.s were less than 2.7%. Analysis took 5 min, making the method suitable for rapid determination of solanesol in N. tabacum. The proposed method has been successfully applied to the analysis of solanesol in various organs of N. tabacum.