In the process of drug development，the bioanalytical is required generally in pharmacokinetic studies. The accuracy，reliability，and scientificity of bioanalytical method validation are crucial，the investigation of matrix effects has become an important part of the validation system for bioanalytical methods. This article investigated related guidelines from ICH，NMPA，FDA and EMA，searching the related domestic and foreign literature on matrix effect，combining with the case study of matrix effect in actual review，to discuss the method of investigating the matrix effect in the methodological verification of drug concentration detection in unknown biological samples，to clarify the causes and solutions of matrix effect. So as to ensure the standardization of the methodological verification of drug concentration detection in unknown biological samples and the accuracy of the test results，to achieve the optimization of drug concentration detection methods for biological samples.