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A feeding trial with 30 dairy cows which were fed rations with three different concentrations of zearalenone (ZEA) and deoxynivalenol (DON) contaminated maize was carried out to examine the ZEA and DON concentration in urine. German Holstein cows (n=30) were divided into three groups (n=10 in each) which received diets with following toxin concentrations: CON (0.02 mg ZEA and 0.07 mg DON, per kg dry matter (DM)), FUS-50 (0.33 mg ZEA and 2.62 mg DON, per kg DM), FUS-100 (0.66 mg ZEA and 5.24 mg DON, per kg DM). For urine analysis, a reliable, cost-efficient and sensitive method for simultaneous determination of ZEA, DON and their metabolites was developed. The method comprises a solid phase extraction clean-up on Oasis HLB cartridges followed by LC-MS/MS measurement. ZEA, alpha-zearalenol, beta-zearalenol, DON and de-epoxydeoxynivalenol (DOM) could be detected in the urine samples of the feeding trial. Thereby, DON was almost completely metabolised to DOM (83-98%) independent of the DON exposure. Moreover, conjugated toxins were the major urinary metabolites based on results of the analysis with beta-glucuronidase treated and untreated samples. Furthermore, relationships between toxin intake and urinary toxin concentration could be established. In conclusion, increased urine toxin concentrations may hint on toxin exposure through the diets and thus the mycotoxins ZEA and DON and their detected metabolites could be used as biomarkers of exposure.