The method was specifically developed for the determination and confirmation of streptomycin in apple samples using the whole mellow apple. The method is simple, rapid, sensitive and was validated for streptomycin in accordance with SANCO/3131/2007. After extraction with phosphate buffer and a pH change, the clean-up was performed by the way of SPE with polymeric phase. The LC–MS/MS analysis was carried out using a HILIC column for the separation of the analytes and a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of streptomycin a matrix calibration curve in the linear range of 1.0–20μgkg −1 and the internal standard dihydrostreptomycin (10μgkg −1 ) were used. The calculated validation parameters like the recovery (101–105%) for 2, 5, 10 and 20μgkg −1 and the relative standard deviation (RSD, 4.1–11.4%) of the 6 replicates fulfil the requirements of SANCO/3131/2007. The LOQ was determined as 2μgkg −1 .