Abstract: The molecular structure and physicochemical property of common aminoglycosides (AGs) antibiotics were systematically analyzed and the residue of animal origin foodstuffs as well as the corresponding damage on human body with long-term intake were introduced. Concrete ideas of the study were raised based on the analysis of present analytical approaches and the discussion of existing defects. An HPLC-MS/MS analytical method was developed for the simultaneous determination of aminoglycosides (spectinomycin, hygromycin B, dihydrostrepmycin, streptomycin, amkacin, kanamycin, apramycin, tobramycin, gentamicin) with a new hydrophilic HPLC column. The residues of aminoglycosides in the test samples were extracted with phosphate buffer solution. After ajusting pH values, the samples were cleaned up with SupelMIP® SPE column, concentrated and reconstituted, the residues were separated by Obelisc R HPLC column (2.1 mm×150 mm×5 μm, 100 ) by gradient elution with acetonitrile-water (1% formic acid) as mobile phase, and detected by liquid chromatography-mass spectrometry under multiple reaction monitoring (MRM) mode via positive-ion mode. All the analytes were calibrated by the external method. The matrix effects were evaluated by comparing solvent prepared standards to matrix-matched standards. The effects of different solid-phase extraction (SPE) columns were optimized. The use of the SupelMIP SPE-Aminoglycoside cartridge kept the analytes bound to the sorbent while a series of aggressive washes were applied to the sorbent to eliminate matrix interferences. The Obelisc R column has reverse phase property and could be applied to conventional reverse phase conditions. It had high density of positive and negative ions on a stationary phase surface, its adsorption capacity in polar compounds could be greatly improved compared with the traditional revers-phase chromatographic column. This allows retaining polar compounds without using ion-pairing reagents. The content of acetonitrile and pH of buffer were adjusted so that the 9 AGs were better retained and separated. The results showed that all the aminoglycosides had good linearity in the range of 20-1 000 μg/L, and the correlation coefficients (R²) were greater than 0.99. The limits of quantification (LOQs) of the nine targets are 50 μg/kg. The average recoveries range from 76.9% to 89.4% for the 9 targets at three spiked levels in pork, and the relative standard deviations were 3.56%-11.4%. This method is proved to be highly sensitive, accurate and reproducible, and it is suitable for the detection of aminoglycosides in pork.