Abstract： Objective An ultra high performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry method (UHPLC-Q Exactive) was developed for the analysis of the related substances in tildipirosin for injection. Methods The UHPLC separation was carried out on a Zorbax SB-C18 (100mm×3.0mm, 1.8μm) by the gradient elution with a mobile phase consisting of 0.1% trifluoroacetic acid aqueous solution (A) and 0.1% trifluoroacetic acid acetonitrile solution (B) at a flow rate of 0.3mL/min. The rapid screening was carried out by Full MS/dd-MS2. The analysis of the related substances were performed with the parent ions and the fragments ions by data dependent scan mode. Results Good resolution of tildipirosin and the main related substances were achieved. Twelve related substances in tildipirosin for injection were separated and detected. Conclusion The established method is effective for the separation and identification of the related substances in tildipirosin for injection and the results are useful for its quality control and process optimization.