Abstract: The fragmentation behavior of Valnemulin was studied in both positive and negative ion mode by electrospray ionization mass spectrometry. The results indicate that the intensity of molecular ion peak M+H⁺ is m/z 565.7 in positive ion mode, and M-H^- is m/z 563.5 in negative ion mode, which response is lower than the former. Then, the M+H⁺ is cracked with suitable collision energy in the MS². The main fragment ions include m/z 263.2, 285.3， 303.3, and the intensity of m/z 263.2 is maximal. This method can be applied for the further research, such as structure-function relationship, pharmaceutical and residual analysis, etc.