Abstract: A method for analyzing sucrose tetraesters (STEs) in tobacco leaves by ultra-performance liquid chromatography-multi-stage mass spectrometry (UPLC-MSⁿ) was developed. The STEs in tobacco samples were extracted by methanol with ultrasonic-assisted extraction under conditions optimized by orthogonal experimental design. The extracts from tobacco samples were cleaned up and then analyzed qualitatively and quantitatively by UPLC-MSⁿ under selected reaction monitoring (SRM) mode. Five groups of STEs were separated and identified by comparing with standard compounds and analyzing the MSn data of the target objects, and the contents of the five groups of STEs were simultaneously and quantitatively determined by using internal standard curves. The results showed that:1) The optimum conditions were solid/liquid ratio 1:30mass (g):volume (mL), ultrasonic power 120 W and extraction time 40 min. 2) The standard recoveries of the quantitative analysis method was in the range of 80.67%-89.35%. The relative standard deviations were less than 5% and the correlation coefficients were no less than 0.999 0. The limits of detection (LODs) and quantitation (LOQs) were 14.71-38.42, 49.02-128.05 μg/kg, respectively. This method is suitable for the quantitative analysis of STEs in tobacco leaves with the advantages of simple pretreatment, higher sensitivity and accuracy.