In the synthesis of larotaxel, a new-generation toxoid, eleven related impurities were detected.In this study, Impurity-I, II, III, IV, VII, IX, X and XI were synthesized, and Impurity-VI, VIII were isolated with the help of preparative high-performance liquid chromatog. (HPLC).The structures of all impurities were characterized using high-resolution mass spectrometry (HRMS) and NMR (NMR) spectral data, and the possible origins of them were explained.Furthermore, a sensitive and accurate HPLC method was developed for the determination of larotaxel and its eleven impurities.The method was validated to fulfill the requirements of the International Conference on Harmonisation (ICH) guidelines, including specificity, sensitivity, precision, accuracy, linearity, and robustness.The validated method can be applied for routine quality control anal. of larotaxel.