Abstract: Objective: To establish the performance verification method of the routine clinical microbial identification system based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and guide clinical laboratories to standardize microbial identification procedures. Methods: One hundred and fifteen strains of standard strains, quality control strains and clinical isolates, including 31 strains of Gram positive/negative cocci, 30 strains of Gram positive/negative bacilli, 30 strains of yeast, 12 strains of anaerobic bacteria and 12 strains of fastidious bacteria, were collected. All strains were verified by the Vitek Compact system, and bacterial 16S rDNA or fungal ITS DNA sequencing. Three MALDI-TOF MS microbial identification systems, including Xiamen mass spectrometry (MS), Bruker MS and Antu MS, were selected randomly. The strains were identified by the recommended method of three detection systems to evaluate their accuracy. Ten strains of standard strains and clinical isolates were identified by one operator using three detection systems for 3 times each day and 3 days, and by three operators using three detection systems for 3 times each day and 3 days, respectively, to evaluate their precision. Results: The accuracy of three detection systems were all 100% for standard strains and quality control strains except anaerobic bacteria, and clinical genus-level isolates. The coincidence rates of Xiamen MS, Bruker MS and Antu MS systems were 100%, 100% and 96.77%, respectively, for species-level Gram positive/negative bacilli, and 96.67%, 96.67% and 100%, respectively, for species-level Gram positive cocci. The coincidence rates of three detection systems were 90% for species-level yeast, 100% for species-level fastidious bacteria, and 91.67% for species-level anaerobic bacteria, respectively. The precision were all 100% for three detection systems. Conclusion: The accuracy and precision of three MALDI-TOF MS systems for the identification of Gram positive/negative cocci, Gram positive/negative bacilli, yeast and fastidious bacteria meet the requirements and pass the verification. The identification of anaerobic bacteria is acceptable at the genus level. The performance verification protocol established in the study can meet the basic requirements of routine microbial identification in clinical microbiology laboratories of general hospitals.