Enzymes as biocatalysts,participate in many physiological processes,which are important biomarkers.The activity analysis of enzymes is of great significance for diagnosis and treatment of many diseases.Matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS) has been widely used in various omics studies and biological molecules detections because of its good performances in sensitivity,throughput,speed and operation.It also has played an important role in enzymes activity analysis.The methods of MALDI-TOF MS for enzyme activity analysis and drug screening are reviewed in this paper,the merits and disadvantages of each method are discussed,the problems and challenges existed are put forward,and finally the perspectives for MALDI-TOF MS in enzyme activity analysis are outlooked.