Abstract: Two pairs of cephalosporins isomers, including Δ₂/Δ₃ isomer of ceftazidime and Z/E isomer of ceftriaxone, were discriminated by electrospray quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS). In addition, the fragmentation characters of cephalosporins were identified by ion trap mass spectrometry. The protonated ceftazidime is found to facilely undergo fragmentation via losing CO or CO₂ to afford the fragment ions at m/z 440, 424 and 396; in contrast, fragmentation of its Δ₃ isomer readily leads to the fragment ion at m/z 313 via the ring-opening reaction initiated by the positive charge on the double bond. For the protonated ceftriaxone, the amide group is facile to interact with the oxygen atom of the oxyimino group via an intramolecular hydrogen bond in the structure of the Z isomer, thereby its fragment ion at m/z 324 shows high abundance. Whereas, there is an intramolecular hydrogen bond between the amide group and the nitrogen atom of the oxyimino group in the structure of its E isomer, which facilitates the loss of methoxyl group to form the fragment ions at m/z 293 and m/z 112. These different fragmentation patterns can be used to discriminate the two pairs of isomers. This work can also contribute to differentiation and characterization of the potential isomeric impurities of cephalosporins.