Introduction: Sedatives have been used for a long time as animal tranquillisers to prevent stress and weight loss during their transportation. The proper determination of these substances in food of animal origin is essential for consumer safety. Material and Methods: A 1 g portion of pig or cow urine or homogenised kidney was mixed with acetonitrile, sodium chloride was added, and the solution was further mixed and then centrifuged. The supernatant was transferred to a new centrifuge tube with primary and secondary amine, octadecylsilane and ZrO2, and mixed rapidly. The filtered solution was evaporated under a nitrogen stream. The residue was dissolved in 200 μL of acetonitrile, centrifuged with filters and then transferred to vials. Samples were analysed by high-performance liquid chromatography–tandem mass spectrometry. Results: The decision limit for confirmation was calculated at 2.5 μg kg−1 for all sedatives with relative standard deviation repeatability and reproducibility below 20%. Conclusion: The validation results showed that this method meets the pertinent EU criteria for such methods and is suitable for sedative analysis in urine and kidney matrices.