Abstract: In source CID of electrospray ionization(ESI)/time of flight mass spectrometry(TOF MS) was applied to five erythromycin analogue antibiotics.Using polyethylene glycol(PEG) and the protonated molecular ion(MH +) as internal reference compounds,the accurate mass of the fragment ions were determined,The fragment ions were elucidated,and their fragmentation patterns were discussed. Erythromycin analogue antibiotics containing structure segments,Desosamine or Cladinose,have more powerful ability to form adduct ions with H + in acid condition.Desosamine can easily form positive ions.The effects of Nozzle Potential and solvents on erythromycin analogue antibiotics of in source CID of electrospray ionization(ESI) were studied.In company with Nozzle Potential rising,the relative abundance of the fragment ions to the protonated molecular ion (MH +) first increases,but after Nozzle Potential reaches to a certain value,it begins to decrease.The higher nozzle potential becomes,the more easily the protonated molecular ion (MH +) methylates with CH 3OH in solvents to form(MH ++CH 3OH-H 2O).And increasing nozzle potential can make the relative abundance of MNa +、MK + rise.Suitable nozzle potential and acid condition were recommended.