Aminoglycosides are a widely used group of antibiotics in veterinary medicine. However, misuse and abuse of these drugs can lead to residues in the edible tissues of animals. Due to thetoxicity of aminoglycosides and the exposure of consumers to the emergence of drug resistance, new methods are being sought to determine aminoglycosides in food. The method presented in thismanuscript describes the determination of twelve aminoglycosides (streptomycin, dihydrostrepto-mycin, spectinomycin, neomycin, gentamicin, hygromycin, paromomycin, kanamycin, tobramycin, amikacin, apramycin, and sisomycin) in thirteen matrices (muscle, kidney, liver, fat, sausages, shrimps, fish honey, milk, eggs, whey powder, sour cream, and curd). Aminoglycosides were isolated from samples with extraction buffer (10 mM NH4OOCH3, 0.4 mM Na2EDTA, 1% NaCl, 2% TCA). For the clean-up purpose, HLB cartridges were used. Analysis was performed using ultra-high-performanceliquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) with a Poroshellanalytical column and a mobile phase of acetonitrile and heptafluorobutyric acid. The method was validated according to Commission Regulation (EU) 2021/808 requirements. Good performance characteristics were obtained for recovery, linearity, precision, specificity, and decision limits (CCα). This simple and high-sensitivity method can determine multi-aminoglycosides in various food samples for confirmatory analysis.