Aminoglycosides are used for disease control, as feed additives, or as synthetic growth promoters within various sectors (livestock farming, aquaculture and agricultural activities) in order to to improve feed utilization and animal production. A high proportion of antibiotics are excreted via urine, feces, milk and eggs as the non-metabolized parent compounds, or accumulate in a high concentration in various tissues. This may pose a real threat to the consumer, either through exposure to the residues, the transfer of antibiotic resistance or increased allergies resulting from antibiotic presence in foods. The negative aspects of the use of antibiotics in animal diets confirm the importance of the development of effective analytical techniques to determine the presence of antibiotics or growth promoters in animal products and the level of bioaccumulation in tissues and in food products. The aim of this study was the determination and quantification of aminoglycosides in samples of milk. For all samples the same procedure was used, so the questioned analytes were extracted threw solid phase extraction (SPE) and were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method has been validated, in accordance with the EU Commission Decision 2002/657/EC and was, threw the use of a softwer program of the instrument MassHunter Quantitative analysis for QQQ version B.07.00, applied to the analysis of aminoglycosides in samples of milk collected from different parts of Croatia.