Abstract: The blood, body fluid and other samples of patients collected by clinical laboratory can be detected to find disease markers through different methods, so as to provide clinicians with an important basis for disease prevention, diagnosis, curative effect and prognosis. LC-MS/MS is an analytical instrument with high sensitivity, high specificity, high resolution and high efficiency. In recent years, with the improvement of instrument sensitivity, LC-MS/MS showed great superiority, practicability and development potential in analytical and clinical practices, thus many diseases can be diagnosed accurately, quickly and sensitively in the early stage. For example, LC-MS/MS was used for neonatal screening, and it could screen more than 30 disease markers including various amino acids and carnitine simultaneously. Comparing with immunoassay, LC-MS/MS applied to endocrine hormones can simultaneously determine a variety of analytes and provide structural information of analytes. It has the advantages of high throughput and high specificity. Due to the expensive instruments and the cost of training, only a few clinical laboratories have the ability to develop and verify LC-MS/MS method. Based on the inherent limitations of the instrument, such as matrix effect and ion suppression effect, the efficiency of the method was decreased, resulting in challenges in the process of method development. In addition, the technical levels of different clinical laboratories are uneven, so it is necessary to establish corresponding reference measurement methods and reference ranges for clinical analysis. The standardization of quantitative results is significant for laboratory to build the traceability system. The research progress of the application of LC-MS/MS in clinical practices was introduced, and the advantages and disadvantages of the application of LC-MS/MS in neonatal screening, vitamin D detection, endocrine hormone detection, therapeutic drug concentration monitoring and protein quantitative analysis were discussed in the review. Finally the possible challenges and the recent advancement that clinical laboratories may confront in the application of LC-MS/MS were discussed.