Abstract: The fragmentation of sodiated molecules M+Na⁺ of five kinds of β-Lactam antibiotics, including three cephalosporins and two penicillins, were studied by electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS). For cephalosporins, besides the cleavage of active tetracycle, 1, 3-H rearrangement and retro-Diels-Alder reaction (RDA) played the substantial roles in the production of many informative product ions, and the D-labeling experiments supported the postulated mechanisms.The processes of sequential losses of a CO₂ and H₂S molecule from the precursor sodiated molecules for penicillins possibly involving 1, 5-H rearrangement were elucidated and further confirmed by D-labeling experiment. This work enriches the fragmentation of β-lactam antibiotics and will be valuable for the rapid identification of untargeted β-lactam antibiotics in the complicated matrix by mass spectrometry.