Abstract: A method of liquid chromatography quadrupole-Orbitrap hybrid mass spectrometry was developed and validated for the determination of ceftiofur and desfuroylceftiofur in chicken. The Kinetex F5 100A column (50 mm×3.0 mm×2.6 μm) was used during the separation of ceftiofur and desfuroylceftiofur. Full MS/dd-MS² mode was chosen for data acquisition, which showed good form in sample qualitative and quantitative analysis. The fragmentation pathways of ceftiofur and desfuroylceftiofur were clarified based on the results of high-accuracy mass spectrometry. During analysis of three types of matrixes (chicken, liver and kidney), correlation coefficients of linear calibration curves of ceftiofur and desfuroylceftiofur are over 0.990 at the corresponding concentration ranges of 2-200 μg/kg. The average recoveries of ceftiofur and desfuroylceftiofur range from 83.2% to 129.7% with the inter-day relative standard deviation (RSD) less than 15% in spiked samples at three levels. Besides, the elimination of ceftiofur in hens was studied. Ceftiofur was injected in hens’ muscle once a day for three consecutive days. The residue can not be detected in chicken breast after 12 h, while ceftiofur and desfuroylceftiofur can be detected in chicken breast simultaneously during 12 h. The metabolism of ceftiofur in hens’ liver and kidney is fast. It can totally metabolize to desfuroylceftiofur during less than 0.5 h. The residue of desfuroylceftiofur in hens’ liver can not be detected after 48 h, while the residue in kidney can not be detected after 72 h. This research can provide a basis for reference for the future development of long plagued quality and safety issues.