Abstract: Destruxins (dtxs) are structurally closely related cyclic hexadepsipeptides secreted asrelevant metabolites by the entomopathogenic fungus Metarhizium anisopliae. To monitor dtxs infungal culture broth, plant derived matrices and cell cultures, a fast and selective off-line SPEUHPLC-DAD/MS method was established. Sample preparation was carried out by a solid phaseextraction (SPE) on a reversed phase material. Optimal purification was achieved by a washingstep with 40% (v/v) methanol, removing most of the polar components. The highest amounts ofdtxs were obtained by using 85% (v/v) methanol for elution. An UHPLC-DAD systemhyphenated to a Q-TOF mass spectrometer was utilized to separate and detect the dtx congeners.A sub-2μm particle size column was used as stationary phase, with a water/acetonitrile solventgradient at a flow rate of 0.3 ml min-1 serving as mobile phase. A total analysis time of 12 minwas achieved with the UHPLC-DAD assay with the dtx congeners eluting from 1 min to 8 minwith a higher resolution of the peaks compared to previous HPLC-DAD assays. Besides theavailable reference compounds dtxA, dtxB, dtxE, dtxE-diol 18 dtx derivatives were tentativelyidentified by analyzing TOF-MS data.