Abstract: In order to clarify the fragmentation behaviors of Sultamicillin (ampicillin/sulbactam), Sultamicillin was investigated by the electrospray ionization  quadrupole  time of flight tandem (ESI-Q-TOF-MS/MS) mass spectrometry in positive ion mode. The fragmentation pathways were elucidated based on the high-resolution of mass spectrometer and isotope-labelling experiment. It was found that the protonated precursor ion M+H⁺ mainly fractured through β-lactam ring of Ampicillin. The M+Na⁺ precursor ion provided more product ions fragmentation by the cleavage of β-lactam, diesters and four-member ring of Sulbactam part. In addition, the loss of neutral molecules such as CO₂, SO₂, CO was occurred readily. The fragmentation experiment of M+H⁺ and M+Na⁺ precursor ions could provide theoretical knowledge for the rapid identification of structural analysis, metabolites in vivo and degradation products in the environment of Sultamicillin.