This protocol describes the untargeted polar analysis of urine samples from cows and calves, with special emphasis on the coverage of thyreostats. Extraction started with 3 mL urine and was in essence based on liquid-liquid extraction with ethyl acetate. Analysis of extracts was achieved by ultra-high performance liquid chromatography (UHPLC) using a Waters Acquity HSS T3 column (1.8 μm, 2.1 x 100 mm), HESI-II ionization, and Q-Exactive Orbitrap mass spectrometry.