Characterization of aminoglycoside antibiotics like ribostamycin is important due to the complex composition and common toxic impurities. Aerosol detectors are often employed for determination of these non-absorbent analytes. In this work, a robust and cost-effective method was developed for simultaneous detection of ribostamycin and its related substances using high-performance liquid chromatography (HPLC) with a relative new aerosol detector named nano-quantity analyte detector (NQAD). With the introduction of less toxic but more compatible ion-pairs pentafluoropropionic acid (PFPA) and trifluoroacetic acid (TFA) in the eluent, an optimized separation effect was achieved. Compared with the other two aerosol detectors namely ELSD (evaporative light scattering detector) and CAD (charged aerosol detector), method verification and quantitative detection results revealed that NQAD had higher sensitivity than ELSD with a 0.8 μg/mL limit of detection, as well as wider linear range (from 2 μg/mL to 1000 μg/mL) than both CAD (from 2 μg/mL to 200 μg/mL) and ELSD (from 8 μg/mL to 200 μg/mL) detector. The performance of NQAD helped to realize detection of ribostamycin and its impurities with significant concentration differences in a single run. With a cation suppressor to eliminate the ion-suppression caused by the ion-pairs in the eluent, the structure of nine impurities in ribostamycin sample was characterized by liquid chromatography-mass spectrum (LC-MS). Both external standard and area normalization calculation were investigated, and NQAD obtained more accurate results due to its full-range linear response-to-concentration relationship, providing an alternative for routine quality control of multi analyte systems. Display omitted •An alternative simple approach for determination of aminoglycoside antibiotics like ribostamycin.•A routine HPLC method for quality control of polar molecules without strong UV absorbing chromophore or fluorophore.•Simultaneous detection of compositions with a wide range of concentrations using the NQAD detector.•Excellent quantification ability in both external standard and area normalization method with the help of NQAD detector.