A rapid, easy, versatile, simultaneous analytical method based on three-layer extraction and LC-MS/MS was developed for the determination of residual veterinary drugs in livestock and fishery products. In this method, degreasing with n-hexane, partitioning into acetonitrile, and purification by the salting-out effect were simultaneously performed after extraction with acetone aq. It allowed to prepare a test solution without concentrating, transferring, and purification using solid column. Recoveries of quinolones were improved by adjusting acid concentration and adding EDTA-2Na・2H2O. As a result, 62 analytes for cattle muscle, 63 analytes for cattle fat, 62 analytes for eel, and 65 analytes for salmon met the management criteria of the guideline for validity assessment (Ministry of Health, Labour and Welfare in Japan). Thus, the present method could be useful for a rapid determination of residual veterinary drugs in livestock and fishery products.