Tuberculosis (TB) is a health public problem and a long combination therapy is necessary to treat patients. In recent years, some drugs, not routinely used in treatment of TB, have appeared as promising new anti-TB therapies in patients with resistance to classical drugs. The aim of this study was: (i) to evaluate a modified checkerboard assay, Resazurin drugs combination microtiter assay (REDCA) to detect drugs interaction in Mycobacterium tuberculosis; (ii) to evaluate the interaction between isoniazid (INH) or ethambutol (EMB) with levofloxacin (LEVO) in susceptible and resistant M. tuberculosis Brazilian clinical isolates. M. tuberculosis H₃₇R_V ATCC 27294 and 19 clinical isolates (10 resistant and 9 susceptible) were tested. The fractional inhibitory concentration index (FICI) ≤ 0.5 was considered synergistic. Synergism in M. tuberculosis H₃₇R_V and resistant M. tuberculosis Brazilian isolates was observed with EMB vs. LEVO. No synergism was observed with INH vs. LEVO by both assays. No statistical difference was observed by the two assays studied. REDCA showed to be a simple assay for detecting synergism between drugs in M. tuberculosis. The results with EMB vs. LEVO are promising and it can be a new option in future investigations of drugs interactions against M. tuberculosis with the view to reduce EMB adverse effects.