Antibiotics are used in the treatment and prevention of animal diseases, and as growth promoters, which can lead to the presence of residues in food of animal origin, which includes milk. According to European Union regulations, MRL (maximum residue limit) for tetracycline antibiotics in milk is 100 μg/kg. The aim of this work was to apply the LC-MS/MS (liquid chromatography tandem mass spectrometry) method and the ELISA (enzyme-linked immunosorbent assay) test, to compare the used extraction methods and to detect their advantages and disadvantages. The optimized parameters of the LC-MS/MS method enabled better selectivity, analyte separation, and more accurate analysis results. By comparing the purification of samples after QuECHERS (quick, easy, cheap, effective, rugged, and safe) and extraction with an organic solvent, it was proven that the extraction yield is higher, and the matrix effect is lower when using extraction with an organic solvent. By using sodium sulfate in relation to magnesium sulfate in QuECHERS extraction, a higher extraction efficiency is achieved. The LCMS/MS method can be used to determine residues of tetracycline antibiotics, while the ELISA test cannot be used for quantitative or qualitative purposes due to the unreproducibility of the results and considering that it does not cover all tetracycline antibiotics that can be found as residues in milk.