It is considered to be difficult to detect tetracycline antibiotics in all-at-once simultaneous analysis with other drugs by liquid chromatography with tandem mass spectrometry, because tetracycline antibiotics chelate with bivalent metal ions such as calcium in samples. Therefore, we studied simultaneous determination of tetracycline antibiotics after removal of calcium with disodium ethylenediaminotetraacetate (EDTA-2Na). Tetracycline antibiotics could be assayed in all-at-once analysis by adding EDTA-2Na during the extraction procedure. It was possible to determine 65 veterinary drugs in milk, 70 in yogurt, 59 in whipped cream, 67 in cheese and 60 in ice cream. Recovery ranged from 70 to 120%, with a coefficient of variation of less than 25% and with a quantification limit of 0.01 microg/g (S/N>or=10).