Abstract: We proposed a liquid–liquid microextraction procedure for the isolation and preconcentration of tetracycline antibiotics from biological fluids. The procedure involves in situ formation of a micellar phase of the primary amine from an aqueous sample solution with the introduction of a polar organic solvent. Possibilities of the developed procedures are demonstrated on an example of the determination of tetracycline, oxytetracycline, and doxycycline in human plasma, blood serum, and urine by HPLC–UV. The limits of detection (3σ) for oxytetracycline and doxycycline were 0.03 mg/L, for tetracycline, it was 0.08 mg/L. The extraction time does not exceed 5 min.