There are more than 160 defined nontuberculous mycobacteria (NTM) species within Mycobacterium genus. In recent years, the number of NTM species associated with human infections and the infections caused by them have been reported at increasing rates. The identification of these species by phenotypic methods is difficult, laborious, and unlikely to obtain reliable results. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) MALDI-TOF MS has proven to be a good method for the identification of bacteria and yeasts in routine laboratory practices. However, Mycobacterium species differ from other bacteria by their cell wall structures, less ribosomal protein content, and slower growth rates. A standardized and efficient protein extraction protocol for MALDI-TOF MS analysis of mycobacteria is essential. The aim of our study was to investigate the efficacy of different protein extraction protocols and the MALDI-TOF MS method in the diagnosis of NTM species. A total of 73 NTM isolates, grown in both solid and liquid media and previously identified with line probe assay, were evaluated with MALDI-TOF MS (Bruker Daltonics GmbH and Co. KG, Germany). Stock isolates were homogenized and decontaminated by N-Acetyl L-cysteine (NALC)/Sodium hydroxide (NaOH) method. For solid media, isolates were inoculated on Löwenstein-Jensen medium and incubated at 35˚C in a normal atmosphere. For liquid media culture, BD BACTEC MGIT 960 automated system (Becton, Dickinson, Sparks, MD, USA) was performed according to the manufacturer's instructions. For the identification of all isolates by MALDI-TOF MS, the manufacturer's recommended protein extraction protocol (Protocol 1) was compared with the two other protocols, using a simplified extraction procedure (Protocol 2), and freezing temperature (Protocol 3). In the liquid media analysis, the rates of the isolates identified by MALDI-TOF MS (score≥ 2.0) for Protocol 1, 2, and 3 were found as 84.93% (n= 62), 63.01% (n= 46), and 43.83% (n= 32), respectively. In the solid media analysis, the rates of the isolates with an identification score of ≥ 2.0 for the protocols with the same order were determined as 87.67% (n= 64), 52.05% (n= 38), and 31.50% (n= 23), respectively. Isolates grown in both solid and liquid media were identified in the same species level in all three protocols, regardless of the identification values and misidentification was not presented. When the reliable identification score was evaluated as ≥ 2.0 in our study, the manufacturer's recommended MYCOEX IVD procedure was found to be the most effective method for the isolates grown in both liquid and solid media. In conclusion, MALDI-TOF MS has the potential to be a reliable, easy-to-use and fast method that can be used in routine practice for the identification of NTM species with its standardized protein extraction protocols.