Abstract: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has the characteristics of high sensitivity, high selectivity and high throughput, and has become the mainstream approach for bioanalytical assays. It is widely used for the quantitative analysis of new drug entities (NCEs) and their metabolites in the discovery and development of new drugs. However, due to the complex composition of the biological sample matrix, the co-eluting material affects the ionization of the analyte, result in increases or decreases the mass spectral response of the analyte, thus affecting the accuracy and precision of the LC-MS/MS analysis method. In general, ion suppression is more common than ion enhancement. When the analytical method is established, if the ion suppression is not evaluated and corrected, the sensitivity of the analytical method will be seriously affected. Therefore, it is important to assess the matrix effect and use different strategies to eliminate or reduce the impact of matrix effects. In this paper, an overview was provided, including how matrix effect occurs, outline the methodologies used to assess the matrix effect in biological sample analysis and discuss the strategy of overcoming the matrix effect. Substances that cause matrix effects include endogenous substances and exogenous substances. Endogenous substances include phospholipid, salts, urea, metabolites and so on. Exogenous substances such as excipients, anticoagulants, stationary phase release materials and degradation products. At present, there are mainly two method to assess the matrix effect, one is post-extraction spike, the other is post-column infusion. The strategies to overcome the matrix effect including the use of stable-isotope labeled internal standard, derivatization of polar drugs, dilute the supernatant after precipitation of the protein, optimize sample pretreatment method, the chromatographic conditions and the mass spectrometry conditions. This paper provides examples of the application of our laboratory, focusing on the difficulties encountered and solutions in the establishment of LC-MS/MS analysis methods.