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The development of an analytical protocol for the identification of the biosynthetic intermediates found in microbial cultures is both challenging and essential for further studies into gene-to-metabolite networks. A highly selective method for detecting the intermediates involved in the nebramycin pathway of S. tenebrarius was developed and validated. The separation of each nebramycin factor through a reversed-phase C18 column using an ion-pairing reagent allowed the simultaneous profiling. By employing the authentic tobramycin spiked into a blank fermentation medium, the combined use of acid extraction, OASIS MCX SPE cleanup, and HFBA-mediated chromatographic separation coupled with ESI-MS/MS detection was proven. The recovery ranged from 89 to 92%, the intra- and inter-day precision (RSD) was <6% and their accuracy ranged from 88 to 93%. A total of nine nebramycin factors including apramycin, 6"-O-carbamoylkanamycin B, 6"-O-carbamoyltobramycin, 3’-hydoxyapramycin, tobramycin, kanamycin B, NK-1012-1, nebramine, and neamine were identified. This is the first report on the integrated LC-ESI-MS/MS characterization of a wide range of nebramycin factors from a bacterial fermentation broth.