Abstract: The purpose of the study was to establish an analytical method to simultaneously determine multiple sterols in tobacco leaves rapidly and accurately. In this gas chromatography-triple quadruple tandem mass spectrometry (GC-MS/MS) based method, various conditions for sterols extraction were assessed and the instrumental operation parameters were optimized with 5α-cholane as an internal standard. Using this method, eight plant sterols, namely lanosterol, campesterol, chenodeoxycholic acid, cholesterol, β-sitosterol, stigmasterol, dihydrotachysterol and ergosterol, were separated in less than 30 min. The linear correlation coefficients were over 0.998 9, the low detection limits were in the range of 0.010 3-0.141 4 μg/g. The recoveries were from 87.30% to 115.60%, with low standard deviations. In conclusion, this method demonstrates good repeatability, accuracy, and high sensitivity, and is best suited for rapid analysis of multiple sterols in tobacco leaves.