Abstract: Polypeptide hormones of tobacco systemin are systemic wound signal, which regulate tobacco growth, development, defense and other activities in the whole life cycle. Therefore it has been desired to develop a sensitive and accurate determination method to find and monitor trace systemins in plants for better understanding molecular mechanisms of polypeptide hormones. In this work, a method of liquid chromatography coupled to linear quadrupole ion trap orbitrap mass spectrometry equipped with an electrospray ionization source (LC-ESI-LTQ Orbitrap-MS) was developed to investigate the fragmentation mechanism of tobacco systemins. The results show that the three-to four-fold protonated systemin ions are observed, and b- and y-type fragment ions are generated at 18-25 V of collision voltages. The optimal collision voltage for M+4H⁴⁺ is found to be 20 V for TobSys Ⅱ, and M+3H³⁺ to be 22 V for TobSys Ⅰ and TobSys Ⅲ. Under the optimized collision voltage, tobacoo systemins have abundant fragment ions and the strongest intensity of the typical fragment ions are b5 (m/z 512.295 3) for TobSys Ⅰ, y12 （m/z 432.218 5） for TobSys Ⅱ, and y8 （m/z 443.712 3） for TobSys Ⅲ, respectively. In addition, the amide of tobacco polypeptide bond to leucine, hydroxyproline and proline are easily cleaved. The method is sensitive and accurate, which is suitable for fragmentation mechanism and qualitative analysis of tobacco polypeptide hormones in tobaccos.