A robust and sensitive method of solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) was established and performed for the simultaneous determination of eleven aminoglycosides (AGs) in royal jelly and honey. After sample extraction by a phosphate buffer containing trichloroacetic acid (TCA) and ethylenediaminetetracetic acid disodium salt (Na₂EDTA), the extraction solution was subjected to a parallel solid-phase extraction for clean-up prior to the LC–MS/MS analysis. The same method was applied to analyze two completely different matrices, honey and royal jelly. Good sensitivity, repeatability, and recovery were obtained by using the mobile phase without an ion-pairing reagent such as heptafluorobutyric acid (HFBA) or sodium heptanesulfonate. The calibration curves of the honey and royal jelly samples exhibited a good linear response (R² > 0.99) at six concentrations in the range of 10–1000 μg/mL. The limit of quantification (LOQ) of the AGs ranged from 10 to 25 μg/kg in the honey and from 12.5 to 25 μg/kg in the royal jelly. The recoveries of the AGs for the honey and royal jelly samples were in the range of 79.48% to 108.95% and 74.61% to 113.70% respectively and the relative standard deviations (RSDs) were between 1.23% and 9.59%, and between 1.51% and 9.98%, respectively. The proposed approach has been allowed in China as a reference method for the simultaneous determination of eleven AGs in honey and royal jelly.