This study aims to establish a rapid and efficient method for qualitative and quantitative determination of plant acid hormones by ultra-high performance liquid chromatography/high resolution time-of-flight mass spectrometry (UPLC- TOF-MS). Several derivatization reagents, 2-bromoacetophenone (BP), 2-dimethylaminoethylamine (DMED), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 3-bromoactonyltrimethylammonium bromide (BTA) were selected for derivatization with abscisic acid, gibberellic acid, indoleacetic acid, jasmonic acid and salicylic acid, respectively. The effect of derivatization reagent was evaluated by comparing the response value of the derivative hormones. The plant hormone standards were diluted in gradient and reacted with the derivatization reagent, and the detection limit and quantitation limit of the derivative hormones were determined. Reagent with the best derivatization effect was reacted with the plant crude extract to test its application effect. All the four derivatized reagents could improve the MS response value of acidic hormone, among which the MS sensitivity of hormone derived by DMED is the best. Reaction with DMED was stable and reproducible. The quantitation limits of ABA-DMED, GA3-DMED, IAA-DMED, JA-DMED and SA-DMED were 0.05, 0.2, 0.1, 0.1 and 0.5 ng∙mL-1, respectively. Compared with non-derivatized hormones, the quantitative limits of ABA, GA3, IAA, JA and SA were reduced by 100, 25, 50, 50 and 10 times, respectively. So, DMED derivatization increased the mass spectrometry sensitivity of several hormones by 10-100 times. The method was applied to the determination of endogenous acidic hormones in rice, wheat and broad bean, and the sensitivity was significantly higher than that before derivation. A simple, rapid and reproducible UPLC-TOF-MS method based on derivatization was established, which greatly improved the sensitivity of the determination of acidic hormones in plants.