A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the quantitative determination of diclazuril in poultry tissues and feed is presented. A simple clean up with an organic solvent was carried out. A reversed-phase C(18) column was used for the high-performance liquid chromatography (HPLC) to separate the analyte with a gradient of acetonitrile and water as mobile phase. The precursor ions produced by electrospray negative ionization were selected for collisional dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC (Off. J. Eur. Communities 2002, L221, 8-36). For the detection of diclazuril in poultry meat, the decision limit was found to be 0.5 microg/kg. An animal experiment was set up in which 70 chickens were held for 6 weeks. From day 22 until day 32, they were fed feed containing 730 microg/kg diclazuril. From day 33 until day 42, every day six chickens were slaughtered, and breast, thigh, and liver were analyzed. Average steady-state concentrations of 94, 135, and 722 microg/kg in breast, thigh, and liver were obtained, respectively. Nine days after withdrawal of the medicated feed, diclazuril was still present in the different sample types.