Simple and sensitive method has been developed and validated for determination of ethambutol (EMB), isoniazid (INH), pyrazinamide (PZA) and rifampicin (RIF) in pure and pharmaceutical dosage forms. The method is based on measuring the quenching effect of studied drugs on the fluorescence intensity of NBS-phenothiazine oxidation product (NBS-Phz) in a buffered medium (pH 7, λex 271 and λem 375 nm). Different factors affecting the reaction were studied and optimized. Under the optimized conditions, linear relationships with good correlation coefficients (0.9995-0.9999) were obtained. The limits of detection were 0.139, 6.39 x 10-3, 0.029 and 0.180 μg ml-1 for EMB, INH, PZA and RIF respectively. The precision of the method was satisfactory with relative standard deviation less than 2 %. Good accuracy was assessed with recovery percentages ranged from 97.24 to 101.2 %. The results were favorably compared with those of the official method. Therefore, the developed method provides applicable tool for determination of studied drugs in pure, pharmaceutical formulation and in quality control laboratories.