In this study, the simultaneous determination of deoxynivalenol (DON), zearalenone (ZEA), T-2 and HT-2 toxins in foodstuff was investigated. A new kind of multi-mycotoxin immunoaffinity columns (IACs) available on the market (DZT MS-PREP®) was tested. A sensitive, selective and accurate method by high-performance liquid chromatography and tandem mass spectrometry was developed, with electrospray ionization mass spectrometer operating in multiple reaction monitoring (MRM) mode, with negative–positive–negative ion switching. The method was used for the analysis of samples marked in Italy, in the frame of official monitoring plans. The advantages of combining IACs and LC–MS/MS technique are as follows: efficient removal of matrix interferences, simple chromatographic outline, high selectivity, low detection limits (DLs) and separation of a wide range of molecules with different physico-chemical properties in a single run. The method was studied on two different matrices, breakfast cereal and baby food, at contamination levels close to Regulation limits (EC) 1126/2007. The recoveries obtained (60–100%) fulfil the performance criteria required by Regulation (EC) 401/2006. The DL is 60 µg/kg for DON and 10 µg/kg for ZEA, T-2 and HT-2. Linearity range of the calibration curves is suitable for adult and baby food. Copyright © 2010 John Wiley & Sons, Ltd.