The objective of the presented study was to develop and optimize a simple, high-throughput method for the control of 32 mycotoxins (Fusarium and Alternaria toxins, aflatoxins, ergot alkaloids, ochratoxins, and sterigmatocystin) in beer. Due to the broad range of their physicochemical properties, the sample preparation step was simplified as much as possible to avoid analyte losses. The addition of acetonitrile to beer samples enabled precipitation of abundant matrix components. The clean-up efficiency was controlled by ambientmass spectrometry employing a direct analysis in real time (DART®) ion source. For determination of analytes, ultra-high-performance liquid chromatography hyphenated with high-resolution mass spectrometry utilizing an orbitrap (U-HPLC–orbitrapMS) or time-of-flight (TOFMS) technology was used. Because of significantly better detection capabilities of the orbitrap technology, the U-HPLC–orbitrapMSmethod was chosen as a determinative step and fully validated. To compensate matrix effects, matrix-matched calibration was employed. The lowest calibration levels for most of the target mycotoxins ranged from 1 to 8mgL-1 beer and the recoveries of analytes were in range from 86 to 124%. Copyright # 2010 John Wiley & Sons, Ltd.