Several streptothricin antibiotics have been studied by tandem mass spectrometry. The dominant product ions were derived from the C(7)-N bond cleavage which lead to lose streptolidine from the [M+H](+). The fragmentation pathways of key ions were described and certained by parent scan. According to the generalized principles, streptothricin isomers could be distinguished easily by the difference of CID spectra. A facile method based on ion-pair RP-HPLC coupled with electrospray ionization tandem mass spectrometry has been established for the analysis of streptothricins in the fermentation broth of Streptomyces qinlingensis. A total of 19 streptothricin-like compounds were identified or tentatively characterized based on their mass spectral data, and in which 11 were the first reported compounds. This could be used as an important de-replication method in the programs of screening novel antibiotics.