Sensitive and reliable analysis of endogenous compounds is critically important for many physiological and pathological studies. Methods based on LC-MS have progressed to become the method of choice for analyzing endogenous compounds. However, the analysis can be challenging due to various factors, including inherent low concentrations in biological samples, low ionization efficiency, undesirable chromatographic behavior and interferences of complex biological. The integration of chemical derivatization with LC-MS could enhance its capabilities in sensitivity and selectivity, and extend its application to a wider range of analytes. In this article, we will review the derivatization strategies in the LC-MS analysis of various endogenous compounds, and provide applications highlighting the impact of these important techniques in the evaluation of pathological events.