Vitamins are a group of compounds that are essential for maintaining good health. As most of them cannot be produced by human body, vitamins are obtained mainly from external sources such as fruits and fish. Analysis methods of HPLC-UV and LC-MS were reported for different types of vitamins. Fat-soluble vitamins are analysed normally using normal-phase chromatography, while water-soluble ones by reversed-phase chromatography. Fish is an important source for both fat-soluble vitamins (D and E) and water-soluble vitamins (B). It is desired to develop methods enabling determination of both fat-soluble and water-soluble vitamins. We describe a novel LC/MS/MS method for simultaneous analysis of 17 fat-soluble and water-soluble vitamins, aiming at quantitation of total vitamins in fish samples using a single method.