Abstract: OBJECTIVE To develop a specific and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the residue determination of spectinomycin, hygromycin B, streptomycin, dihydrostreptomycin and amikacin in honey. METHODS After extraction of these analytes from honey samples with a phosphate buffer, the extract was loaded on a weak cation-exchange (WCX) solid-phase extraction column. After sample preparation, the hydrophilic interaction chromatography (HILIC) was used to retain the aminoglycosides on the column for the separation of the analytes from endogenous compounds. The aminoglycosides were detected and quantified with triple quadrupole tandem mass spectrometry with ESI source in the positive ion mode. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions with isepamycin as the internal standard. RESULTS The linear regressions were fitted over the concentration range of 10-1 000 μg·kg^-1 for the analytes in honey. The lower limit of quantification of the analytes were all in 0.6-5.2 μg·kg^-1. The method and extracted recoveries of the analytes evaluated by determining the spiked samples at all levels were 92.4%-103.2% and 62.5%-81.6%, respectively. CONCLUSION The developed LC-MS/MS method is accurate and efficient enough to determine aminoglycosides in honey.