Abstract: In order to determine acidic phytohormones in tobacco fast and accurately, an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for detecting abscisic acid(ABA) and jasmonic acid(JA) in tobacco was developed by improving the conditions of sample pretreatment. The results showed that: 1) The detection limits of ABA and JA were 0.082 and 0.027 ng/m L, respectively; their standard addition recoveries ranged from96.4% to 110.8%. The intra- and inter-day relative standard deviations(RSDs) of ABA were less than 3.86%, and those of JA were less than 4.53%. 2) Only 25 mg of tobacco sample were needed for detection; and the negative ion detection mode adopted by this method featured high selectivity and less interference. This method provides a basis for accurately detecting the endogenous levels of acidic phytohormones.