A method was specifically developed for the determination and confirmation of streptomycin and dihydrostreptomycin in different types of honey. The method is simple, rapid, sensitive and was validated for streptomycin and dihydrostreptomycin in accordance with Commission Decision 2002/657/EC. After extraction with phosphate buffer and a pH change, clean-up was performed via SPE with polymeric phase. LC-MS/MS analysis was carried out using two different HILIC columns for the separation of the analytes and using a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of both substances, matrix calibration curves in a linear range of 5-80 g kg(-1) were used. The validation parameters established for streptomycin and dihydrostreptomycin, CCα (11.8 and 11.5 µg kg(-1), respectively), CCβ (18.9 and 19.9 µg kg(-1), respectively), recovery (97 and 101%, respectively) and the relative within-laboratory reproducibility RSD(wR) (16.4 and 20.8%, respectively) at the recommended concentration of 40 µg kg(-1), fulfil the requirements of Commission Decision 2002/657/EC.