Abstract: A method of high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established for determining of maleic hydrazide residue in tobacco. The LC separation was performed using water containing 0.1% formic acid and methanol containing 0.1% formic acid as mobile phases with gradient elution at a flow rate of 0.3 mL/min. Qualitative and quantitative analysis were achieved after the chromatographic separation on a Thermo Hypercarb column (100 mm×2.1 mm×5 μm). The ESI source in positive ion mode was used for analyzing maleic hydrazide in the multiple reaction monitoring (MRM) mode. The pretreatment parameters were optimized, such as the extraction methods, the concentration and volume of extracts. The tobacco samples were extracted by 40 mL HCl aqueous solution at 2 mol/L under heat reflux for 1 h with d₂-maleic hydrazide as the internal standard. After dilution with water and filtration by 0.45 μm membrane filters, the obtained solution was analyzed by LC-ESI-MS/MS in positive MRM mode. The results show that the standard curve is obtained with the correlation coefficients (R²=0.999 9) in the concentration range of 1-100 mg/kg. The limit of quantitation (LOQ, S/N=10) for maleic hydrazide is 0.27 mg/kg. The recoveries of maleic hydrazide are 90.3%-101.5% at three spiked levels of 2, 10 and 50 mg/kg with the relative standard deviations (RSDs) of 2.6%-5.5% (n=6). 12 tobacco samples were selected to detect, in which the maleic hydrazide were not detected in 10 samples. For the rest of samples, in comparison the results of this method with the existing standard method, there was no significant difference. This method is simple and accurate, which is suitable for the determination of maleic hydrazide in tobacco.