A precise liquid chromatographic (LC) determination with high sensitivity and a reliable mass spectrometric (MS) confirmation were established for the determination of tetracycline antibiotics (TCs) in milk. The determination of the TCs was based on C18 cartridge cleanup followed by LC separation. Recoveries of TCs from milk fortified at 20 ppb were 73.0-81.8%, and coefficients of variation were 2.6-4.6%. With this method, concentrations of oxytetracycline and tetracycline as low as 10 ppb and chlortetracycline and doxycycline as low as 20 ppb were determined readily. Thin-layer chromatography (TLC)/fast atom bombardment (FAB) MS with a condensation technique was used to confirm TCs in milk. After the C18 cartridge cleanup, separation on a C8 TLC plate, and application of the sample condensation technique, FAB mass spectra were measured. TCs in milk at 50 ppb were reliably confirmed by TLC/FABMS.