This work aimed to develop an analytical method for the screening of multiple aminoglycoside residues in foods of animal origin using an ethylene-bridged hybrid (BEH) particle-based sulfoalkylbetaine stationary phase. The effects of chromatographic conditions on the separation of 17 aminoglycosides have been systematically investigated. Sample preparation and mass spectrometry detection have also been investigated and optimized. In contrast to high buffer concentrations in the mobile phase required for silica-based sulfoalkylbetaine stationary phases, a moderate buffer concentration (20 mM) provided the optimal separation of 17 aminoglycosides with the BEH sulfoalkylbetaine stationary phase. The developed method has been evaluated in milk, beef, pork, liver, and honey samples with good performance for retention, selectivity, sensitivity, linearity, precision, and accuracy. The majority of the limit of quantitation estimated with the matrix was less than 25 μg/kg. The overall accuracy across five matrices was in the range from 96 to 111%, with standard deviations of less than 19%.