The main aim of this work was to determine the effect of high pasteurization of milk (85 °C/3 s) on the residues of tetracycline and oxytetracycline. The samples of raw cow’s milk, purchased from a vending machine, were spiked with standard solutions of tetracycline and oxytetracycline. The content of the residues of tetracycline antibiotics was measured before and after heating. Pre-cleaned samples were extracted by a mixed-mode solid phase extraction technique and analysed using high performance liquid chromatography/diode array detection. Whereas the residues of tetracycline decreased only by 5.74% and were not significantly different (P > 0.05), the residues of oxytetracycline decreased by 15.3% and this distinction was highly significant (P ≤ 0.01). Based on the results of our study, the tetracycline antibiotics were proved to have differences in the thermostability of particular substances at pasteurisation temperatures.