<jats:p>A high‐performance liquid chromatography with electrospray ionization ion trap tandem mass spectrometry method was developed and validated for the robust profiling and characterization of biosynthetic congeners in the 2‐deoxy‐aminocyclitol istamycin pathway, from the fermentation broth of <jats:italic>Streptomyces tenjimariensis</jats:italic> ATCC 31603. Gradient elution on an Acquity CSH C<jats:sub>18</jats:sub> column was performed with a gradient of 5 mM aqueous pentafluoropropionic acid and 50% acetonitrile. Sixteen natural istamycin congeners were profiled and quantified in descending order; istamycin A, istamycin B, istamycin A<jats:sub>0</jats:sub>, istamycin B<jats:sub>0</jats:sub>, istamycin B<jats:sub>1</jats:sub>, istamycin A<jats:sub>1</jats:sub>, istamycin C, istamycin A<jats:sub>2</jats:sub>, istamycin C<jats:sub>1</jats:sub>, istamycin C<jats:sub>0</jats:sub>, istamycin X<jats:sub>0</jats:sub>, istamycin A<jats:sub>3</jats:sub>, istamycin Y<jats:sub>0</jats:sub>, istamycin B<jats:sub>3</jats:sub>, and istamycin FU‐10 plus istamycin AP. In addition, a total of five sets of 1‐ or 3‐epimeric pairs were chromatographically separated using a macrocyclic glycopeptide‐bonded chiral column. The lower limit of quantification of istamycin‐A present in <jats:italic>S. tenjimariensis</jats:italic> fermentation was estimated to be 2.2 ng/mL. The simultaneous identification of a wide range of 2‐deoxy‐aminocyclitol‐type istamycin profiles from bacterial fermentation was determined for the first time by employing high‐performance liquid chromatography with tandem mass spectrometry analysis and the separation of istamycin epimers.