Abstract <p>A polymethacrylate matrix (<b>PMM</b>) is proposed for the solid-phase extraction and determination of tetracycline in water and human urine. A study of the effects of medium acidity, temperature, and contact time on tetracycline extraction by PMM demonstrates that tetracycline is extracted by the matrix as the singly charged anion H₂TC⁻ in the pH range 8.9–9.7, with partition coefficients reaching (5–6) × 10³ mL/g. The sensitivity of the solid-phase spectrophotometric determination of tetracycline after its extraction with PMM is increased by heating analyte solutions to 60–70°C at a contact time of 60–120 min. A procedure for the solid-phase spectrophotometric determination of tetracycline with a limit of detection of 0.03 mg/L after extraction of tetracycline for 90 min is developed. Using the instrumentally measured peak area of the anionic form of tetracycline H₂TC⁻ in the matrix at the absorption maximum 385 nm as an analytical signal reduces the limit of detection to 0.01 mg/L. The procedure was tested in the analysis of river and bottled water samples and human urine.</p>