Rapid and accurate identification of non-tuberculous mycobacteria (NTM) is important for a prompt start to antibiotic therapy. The aim of this study was to obtain accurate identification of NTM quickly by analyzing the performance of the MALDI-TOF mass spectrometry (MS) system VITEK® MS in identifying various NTM species from solid medium and MGIT 960 liquid medium. The study was performed in two phases: preliminary and perspective. Overall, 41/42 species and 33/34 species were correctly identified from the MGIT medium in the preliminary and perspective phases, respectively. The VITEK® MS system includes in its database part of the mycobacteria from the Mycobacterium fortuitum complex but is unable to discriminate among the various species belonging to the complex. Although the VITEK® MS system does not have the protein spectrum of Mycobacterium chimaera, it is not able to distinguish between Mycobacterium chimaera and Mycobacterium intracellulare. Since the VITEK® MS includes the separate protein spectrum of both M. chelonae and M. abscessus, it can discriminate between the two microorganisms. Thanks to these studies we show that the VITEK® MS system is a reliable method for identification of NTMs directly from MGIT liquid medium, instead of the use of solid media.