Background: Matrix-assisted laser desorption ionization- time of flight mass spectrometry (MALDI-TOF MS) has recently been used in microbiology laboratories for rapid and accurate identification of bacterial and yeast strains isolated from clinical samples. Objective: To evaluate the analytical and practical performance of MALDI -TOF MS (VILEK MS) system in comparison to conventional VITEK2 system for identification of aerobic gram positive and yeast strains. Methodology: A total of 416 isolates (340 aerobic Gram positive bacteria and 76 yeast strains) representing 5 genera and 30 species were analysed. Unmatched results were further investigated by 16s rRNA gene sequencing according to a reference method. Results: For 416 isolates, All strains (100%) were accurately identified at the genus level and 409 (98.3%) isolates at species level by using MALDI TOF MS. A total of 388 (93.26%) isolates were completely matched by both methods. There were 28 unmatched isolates, MALDI TOF MS made no errors at the genus level while VITEK 2 made 4 (0.96%) errors at the same level. At the species level the identification error rates of MALDI -TOF MS and VITEK 2 were 1.68% and 5.04% respectively. Conclusion: our findings demonstrated that MALDI -TOF MS has better performance than VITEK 2 in microbial identification. It is accurate, quick and cost- effective technique for bacterial identification in the Clinical Microbiology Laboratory.