Abstract: Derivatization, which involves transforming target analytes into derivatives that are more amenable to separation and ionization, can significantly enhance the separation, sensitivity, and/or selectivity for detecting trace compounds in complex matrices. The method has been extensively applied in LC-MS analysis.This review introduces the fundamental principles and advantages of derivatization, as well as its applications in sample pretreatment. This review also summarizes the different types of chemical derivatization reactions and their specific applications in fields such as pharmaceuticals, food, life sciences, and environmental analysis. Additionally, the advancements in stable isotope labeling derivatization and enzymatic derivatization are explored through emphasizing the importance of derivatization in enhancing the sensitivity, selectivity, and accuracy of LC–MS analysis. Overall, derivatization has become a significant branch in the field of LC-MS analysis, demonstrating broad application prospects.