Abstract: An SFC-MS-MS method was optimized and established for the simultaneous, accurate and rapid determination of free lipid components in tobacco. Through systematic optimization of sample-pretreatment conditions and instrumental parameters, including column type, mobile phase, modifier, ion source, etc., 15 lipid components were separated efficiently and determined, involving α-cembra-trienediol, β-cembratrienediol, α-tocopherol, α-tocotrienol, β-sitosterol, campesterol, brassicasterol, cholesterol, stigmasterol, ergosterol, lanosterol, squalene, labd-13-ene-8,15-diol, cis-abienol and solanesol. The results showed that: 1) All analytes exhibited good linearity within the tested ranges, with all of the correlation coefficients (r) more than 0.99. 2) Recoveries at different spiked levels ranged from 81.6% to 114.9%. Intra- and inter-day RSDs were 1.79%–6.29% and 1.74%–7.29%, respectively. Limits of detection (LODs) and limits of quantification (LOQs) were 0.000 87–0.26 and 0.002 9–0.85 μg/mL, respectively. 3) Application of this method to various tobacco samples showed that this method could successfully detect the target lipid components in different types of tobacco sample. Compared with the traditional methods, this method exhibited strong matrix compatibility, high separation efficiency and sensitivity. It reduced organic solvent consumption and was eco-friendly. It could achieve simultaneous, accurate and rapid quantitative determination of many free lipid components in tobacco.