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A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 µg/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 µg/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.