The aim of this study was to develop a high-precision methodology that would allow the detection and quantification of tetracycline residues in cheeses using the Liquid Chromatography High Efficiency (HPLC) analytical technique. As a result, a method was generated in which tetracycline residues were detected in complex biological matrices such as cheese. Conditions of temperature (30 ° C), wave length (265 nanometres (nm)), volume (20 microlitres (μL) and injection flow (1 millilitres mL / minutes (min)) were established, as well as the mobile phase used with methanol: acetonitrile: oxalic acid (74 : 13: 13% volume/ volume (v/v)) for the assay on the HPLC machine. Calibration curves with coefficient of determination (R2) close to 1 were obtained, with concentrations of standard solutions of 2.0 parts per million (ppm), 1.5 ppm, 1.0 ppm, 0.8 ppm, 0.6 ppm, 0.5 ppm, for tetracycline. The method presented quantification limits for tetracycline of 0.1 milligrams/Litter (mg/L) and detection limit of 0.02 mg/L, values that are below the maximum limits allowed by the Colombian standard for milk. On the other hand, conditions were established to carry out the extraction process of the antibiotic residues in complex biological matrices, such as buffer, centrifugation time and temperature, type of cartridge for purification and recovery reagent, among others. It is concluded that the recovery percentage was adequate, and it is evident that the method allows the detection of antibiotic residues from biological samples.