Abstract: Fluoroquinolones (FQs) are the third generation of quinolones (QNs), which are one of the most important breakthrough in the field of artificially synthesis of antimicrobial agents following sulfa drugs. Because of its wide antimicrobial spectrum, strong antibacterial activity, no cross resistance side effects with other antimicrobial agents, FQs are widely used in small animal and human many kinds of infectious diseases prevention and treatment. However, with the widespread use of antibiotics, the problems of counterfeit antibiotics emerged and proliferated gradually. Therefore, it is need to develope an analytical method with high sensitive and high speed. Mass spectrometry was the development of ambient ionization techniques, which enabled the ionization of samples in their native environment without sample pretreatment. After the emergence of the pioneered technique, desorption electrospray ionization (DESI), including more than 20 direct-ionization techniques, have been developed in the past decades. Among them, desorption atmospheric pressure chemical ionization (DAPCI) is a powerful ionization technique and has been successfully applied to detect trace surface analytes without evidently injuring the sample in food security, forensic science and environmental science. In this study, the DAPCI source coupled with the linear ion trap mass spectrometer was used to investigate the fragmentation mechanism of fluroquinolones. Five compounds of fluroquinolones were analyzed using surface desorption atomospheric pressure chemical ionization mass spectrometry (DAPCI-MS) by collision induced dissociation in positive ion detection mode. The mass spectra and structures of the five fluroquinolones were compared with each other, and it was observed that fluroquinolones gave characteristic fragment ions by the neutral loss of 44 u (CO₂), 28 u (CO), 20 u (HF) and 18 u (H₂O). If compounds have piperazidine substituent, piperazine-link rearrangement can be observed after decarboxyliation, and characteristic fragment ions by the loss of the 43 u (CH₂CDS1CH—NH₂) and 57 u (CH₃—CH₂—NCDS1CH₂) were produced. All of them can be used as a “diagnosis” of other fluoroquinolone compounds and structure similar to the characteristics of the ions, which can open a way to fast analyze the FQ drugs.