Abstract: Colistin is a kind of polypeptide antibiotic, which is used for the treatment of infection caused by gram-negative bacteria. The market colistin is usually the mixture of colistin A and colistin B, and these two components show similar antibiotic activity. However, it was reported that colistin A causes much more damage to kidney than colistin B. In addition, colistin methanesulfonate is inactive prodrug of colistin, which is metabolized to colistin in vivo to kill bacteria. Colistin B methanesulfonate is a safer and more active drug compared with the mixture. There are many researches about colistin and colistin methanesulfonate. However, most of researches for colistin methanesulfonate are indirect. It is necessary to develop a method to directly study colistin B methanesulfonate. In this study, ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF MS) was used to study colistin B methanesulfonate. Acquity UPLC HSS T3 column (100 mm×2.1 mm×1.8 μm) and gradient elution were selected for better separation of colistin B with different numbers of methanesulfonic acid groups. The mass detection method for analysis of collision induced dissociation (CID) of colistin B with different numbers of methanesulfonic acid groups and deducing the possible fragmentation pathways was established. It was concluded that the retention time of colistin B with different numbers of methanesulfonic acid groups depended on the number of methanesulfonic acid groups. The more numbers of methanesulfonic acid groups were, the shorter retention time was. In addition, colistin B with different numbers of methanesulfonic acid groups could be separated well through gradient elution but isomers of the same number of methanesulfonic acid groups couldn’t be separated well because of the similar property of these isomers. It was revealed that colistin B with different numbers of methanesulfonic acid groups all had double charge quasi-molecular ion peak M-2H^2-. CID research showed that CH₂SO₃, H₂SO₃, SO₂, CH₃CHO were the main neutral fragments without any amide bonds cleavage. This study provides a reference for the further study of the mass spectrometry of colistin B methanesulfonate and its derivatives, and also has a reference for the quantitative analysis and pharmacokinetic study of colistin B methanesulfonate.