A method was established for simultaneous determination of 16 aminoglycoside (AG) residues in beef. Samples were extracted with phosphate buffered solution and the proteins were precipitated using trichloroacetic acid. After adjusting the pH, the target compounds were cleaned up on an HLB solid phase extraction (SPE) column, separated on a Hilic Plus column, and detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in the positive ion electrospray ionization mode. The results showed that the calibration curves for the 16 AGs exhibited good linearity in the concentration range of 25–750 μg/kg with correlation coefficients greater than 0.990. The limits of detection (LOD) were 0.006–0.609 µg/kg and the limits of quantitation (LOQ) were 0.02–1.83 µg/kg. The average recoveries at three spiked levels were in the range of 69.09%–115.50%, with relative standard deviations (RSDs) of 1.04%–16.58% (n = 6). The method is sensitive, accurate, and suitable for simultaneous determination of the 16 AGs in beef.