Introduction: Phenotypic methods for drug susceptibility testing of Mycobacterium tuberculosis complex (MTC) to second-line drugs are not yet standardized. The Sensititre MycoTB Plate is a microtiter plate containing lyophilized antibiotics and configured for determination of MIC to first and second-line anti-tuberculosis drugs. The purpose of this study is to detect the susceptibility rates of MTC strains isolated from patients’ specimens for first and second-line anti-tuberculosis drugs. Materials and Methods: This study included 50 MTC strains isolated from various clinical specimens. Out of the 50 strains, 38 were isolated from sputum, three from cerebrospinal fluid, three from bronchoalveolar lavage, and six from other samples in this study. The susceptibility of strains to anti-tuberculosis drugs were determined by the Sensititre MycoTB Plate Method. Thawed isolates were subcultured, and dilutions were inoculated into MycoTB wells. The results were read at days 7, 14 and 21. Results: At the end of study, out of 50 MTC isolates, 7 (14%) showed resistance to Isoniazid (INH), 5 (10%) to streptomycin (SM), 4 (8%) to ethambutol (EMB), 4 (8%) to ethionamide (ETH), 3 (6%) to rifampicin (RIF), 3 (6%) to rifabutin (RFB), 2 (4%) to kanamycin (KAN), 2 (4%) to ofloxacin (OFL), 2 (4%) to P-aminosalicyclic acid (PAS), 1 (2%) to moxiflocacin (MOX), and 1 (2%) to cycloserine (CYC). All strains were found sensitive to amikacin while 2 strains (4%) were identified as multidrug-resistant tuberculosis (MDR-TB). Thirty-five strains (70%) were sensitive to all drugs. Extensively drug resistant tuberculosis (XDR-TB) was not determined in this study. Conclusion: This is the first study that tests second line anti-tuberculosis drugs in our location and provides us valuable data regarding MDR-TB and XDR-TB rates. The Sensititre MycoTB Plate Method is a fast, reliable and practical method and can be used to determine the susceptibility of first and second-line anti-tuberculosis drugs.