10-O-4-Ketonephenyl carbamate docetaxel (DTX-AI) is a synthetic taxane compound that exerts antitumor effects by inhibiting microtubule depolymerization and promoting microtubule dimer synthesis. Owing to the poor water solubility of DTX-AI, the liposomal formulation for injection was developed and prepared. In vitro experiments showed that DTX-AI and its liposomal formulation outperformed DTX in terms of antitumor efficacy at much lower concentrations, with correspondingly lower toxicity. The quality and stability as well as liposomal formulation of DTX-AI directly impact its therapeutic efficacy and safety. This study utilized LC-QTOF-HRMS to isolate and identify the process and stress testing degradation related substances (RSs) in DTX-AI. A total of 23 RSs were detected and identified in the Active pharmaceutical ingredient (API) via positive ESI-HRMS, and their structures and degradation pathways were elucidated and summarized. These findings provide valuable insights into the optimal production processes, formulations, storage conditions, and quality control for DTX-AI.