Aminoglycosides are broad-spectrum antibiotics often employed to combat Gram-negative bacterial infections. A technique based on electrospray-ionization mass spectrometry (ESI-MS) was developed for rapid determination of aminoglycosides. This method, which does not require prior chromatographic separation, or derivatization and extensive sample preparation steps, was deployed to estimate gentamicin, tobramycin, and amikacin in pharmaceutical formulations. Upon gas-phase collisional activation, protonated gentamicin, tobramycin, and amikacin undergo a facile loss of their respective “C” ring moiety to produce characteristic ions of
m/z322, 324, and 425, respectively. The mass spectral peak intensities for these specific product ions were monitored either by a flow-injection analysis selected-ion monitoring (FIA-SIM) time-intensity method or by a mass spectrometric internal-standard method. The linear dynamic ranges of detection for both methods were evaluated to be 10–1000 ng/mL for gentamicin, 25–2500 ng/mL for tobramycin, and 10–1000 ng/mL for amikacin. The internal-standard mass spectrometric method afforded lower intra-day and inter-day variations (2.3–3.0% RSD) compared to those from FIA-SIM method (4.5–5.0% RSD). This method was applied as a potential alternative procedure to determine gentamicin in commercial pharmaceutical samples and to monitor the release of gentamicin from “self-defensive” tannic acid-based layer-by-layer films into phosphate buffer solutions at different pHs.