Ceftiofur is a third-generation cephalosporin antibiotic used to treat cattle and swine for bacterial infection of the respiratory tract. It is not authorised for use in poultry within the European Union. Due to the complexity of the chemistry and metabolism of ceftiofur, maximum residue limits (MRLs) are based on desfuroylceftiofur (DFC) equivalents after chemical conversion of all compounds that have an intact ß-lactam ring. In practice the DFC is usually stabilised as the acetamide (desfuroylceftiofur acetamide - DFCA) for analysis. Because of recent evidence of off-label use in the European Union, a policy need emerged to develop a cost-effective method for the detection of ceftiofur residues in poultry tissues. One-day-old chicks were each dosed subcutaneously with ceftiofur and samples taken from day 1 to day 44 post-dosing. Residues of ceftiofur parent compound were detected in whole chicks, wing feathers and faeces. On the basis of this finding it was decided to evaluate ceftiofur parent, as the marker, instead of proceeding with the time-consuming conversion to DFCA. Expected metabolites, DFC and desfuroylceftiofur cysteine disulfide (DCCD), were not detected in whole chicks, muscle or liver, but DFC was found in wing feathers. These results indicate that determination of ceftiofur parent compound in either whole chicks or possibly wing feathers and faeces may allow the detection of the misuse of ceftiofur.