ABSTRACT Oxytetracycline (OTC) is used for the prophylaxis and treatment of a great number of diseases since this antibiotic possesses broad-spectrum activity against many pathogenic organisms. The use of OTC has become a serious problem because of the possible existence of its residues in milk, which can be directly toxic or cause allergic reactions in some hypersensitive individuals. Even low-level doses of antibiotic in milk consumed for long periods can lead to problems regarding the spread of drug-resistant microorganisms. The purpose of the present study was to investigate residual OTC in consuming milk in Tehran using high-performance liquid chromatography (HPLC) with UV detector. OTC residues in extracts obtained from a preliminary cleanup procedure and recoveries from spiked OTC in desire concentrations were between 80% and 97% with appropriate coefficients of variation. The limit of detection (LOD) and limit of determination (LOQ) were 50 and 68.5 ng/mL, respectively. This result shows that this method would be useful for routine monitoring of oxytetracycline residues in bovine dairy milk.