Thiouracil (TU) is a representative of a group of thyreostatics, orally active drugs that can be used to increase the weight of cattle before slaughter. The use of thyreostats in animal production has been banned in the European Union since 1981. Systematic detection of low concentrations of thiouracil in the urine of livestock is acknowledged to be of endogenous origin due to the use of Brassicaceae plants in the animal diet. The purpose of the study was to determine the effect of diet enriched with rapeseed meal on endogenous thiouracil contents in urine of calves. Two groups of calves of different age were subjected to a rapeseed-supplemented diet. First group (n = 6, 6 months) received a maximum authorized content of rapeseed meal (30%) in diet, second (n = 2, 2 months) after adaptation to solid food and rapeseed, has been subjected to a diet exceeding the maximum recommended dose of rapeseed of 10%. During the experiments, samples of urine and faeces were collected and analyzed by using the accredited LC-MS/MS method. The diet with allowed content of rapeseed did not influenced on concentration of endogenous thiouracil in calves' urine (<CCα – 3.11 μg/L), while exceeding the permitted dose caused a significant increase in thiouracil in urine of younger animals (1.55–20.95 μg/L) and there may be a reason for non-compliant results. The higher levels of endogenous thiouracil observed in young calves results from exceeding the recommended dose of rapeseed in diet as well as from intensive feeding of animals during growth.