Abstract: Streptomycin (STR) and dihydrostreptomycin (DSTR) are two of the most common aminoglycoside antibiotics used in veterinary medicine. STR is produced by some streptomyces griseus strains, and DSTR is a derivative of STR. In recent years, STR has been widely used in grapes to induce denuclearization. However, high levels of STR may have adverse effects like serious ototoxicity and nephrotoxicity. Therefore, to ensure the quality of grapes and the health of consumers, the regulation of STR and DSTR levels in grapes is required. An analytical method was developed for the identification and quantification of STR and DSTR in grapes by liquid chromatography-tandem mass spectrometry (LC-MS/MS). STR and DSTR are highly polar compounds due to the presence of various amino and hydroxyl groups in their structure. The determination of STR and DSTR poses a considerable analytical challenge, both during sample preparation and instrument analysis. In this study, the main factors governing the response, recovery, and sensitivity of these compounds, such as the type of chromatographic column, the type and proportion of the mobile phase and extraction solvent, the dosage of sodium 1-hexane sulfonate solution, and elution solvent and its volume, were investigated during sample pretreatment and instrument analysis. The STR and DSTR residues in the grape sample were extracted by ultrasonication with a phosphoric acid solution (pH 2), and cleanup and enrichment was performed using an Oasis HLB solid phase column. The analysis was performed using a UPLC Waters HSS T3 column (100 mm×2.1 mm, 1.8 μm) at the column temperature of 35℃. The injection volume was 2 μL. The mobile phase consisted of 0.1% formic acid aqueous solution and methanol with a volume ratio of 60:40. ESI-MS/MS was operated in multiple reaction monitoring (MRM) mode. External standard calibration curves were used for quantification. Based on the optimized method, both analytes displayed good linearity between 2 and 400 μg/L. The correlation coefficients were 0.9991-0.9997. Recoveries in spiked blank grape samples (5, 10, 20, and 40 μg/kg) ranged from 76.8% to 91.9%, with the relative standard deviations (RSDs) less than 10.2%, in compliance with the current legislation. The limits of detection and the limits of quantification of both analytes were 1 μg/L and 5 μg/kg, respectively. To assess the feasibility and potential of the proposed approach for routine analyses of STR and DSTR in other kinds of grape samples, the developed method was applied to the analysis of these compounds in red grapes, xinyu grapes, and xiahei grapes. The recoveries of STR and DSTR in the three kinds of blank grape samples were 77.2%-83.9% and 70.8%-78.9%, respectively, and the RSDs ranged from 3.0% to 15.6%. The results showed that the optimized methods can yield satisfactory recoveries for the analytes in grapes. In this method, the combination of Waters HSS T3 column to overcome the difficulties of the retention and separation of these highly polar compounds in the reverse phase, avoids the use of an ion-pair additive in the mobile phase to increase their retention, which is known to cause severe contamination of the column and serious ion suppression with electrospray ionization detection. In addition, the ideal enrichment and purification effect can be achieved by adding a sodium 1-hexane sulfonate solution to the superstratum extract with the use of only Oasis HLB for sample treatment. The method described herein has the advantages of easy operation, accuracy, and selectivity, making it feasible for the identification and quantification of STR and DSTR residues in grapes.