Protein polypeptides are a class of bioactive substances that play a crucial role in maintaining the stability of various functions of the organism. Rapid and accurate detection of their levels could help in the diagnosis of diseases, the monitoring of drug therapy and the research and development of medicines, which is of great significance in the fields of clinical medicine, biology and pharmacy. Conventional protein polypeptides detection methods, such as Western blotting and enzyme-linked immunosorbent assay, still have problems like low sensitivity or difficulty in determining more than 1 analyte simultaneously. Liquid chromatography-mass spectrometry (LC-MS) has the advantages of specificity, sensitivity and high throughput. However, low ionization efficiency of macromolecules and strong biological matrix effects limit the feasibility of direct detection of protein polypeptides by LC-MS, which has led to the development of signal conversion and amplification strategies based on LC-MS technology. In this review, we summarized the research progress of sample pretreatment methods and signal conversion and amplification strategies for quantification of protein polypeptides in vivo based on LC-MS in recent years, providing a reference for developing specific high-sensitivity detection methods for low-abundance protein polypeptides in complex samples based on LC-MS technology.