We report the applicability of testing susceptibility to paranitrobenzoic acid (PNB) directly on clinical samples as a rapid screening assay, to detect M. tuberculosis and differentiate it from non-tuberculous mycobacteria (NTM). One hundred smear positive sputum samples from patients with pulmonary tuberculosis attending the Department of Respiratory Medicine at VP Chest Institute, Delhi, were cultured on Löwenstein Jensen medium with and without 0.5 mg/ml paranitrobenzoic acid. Serial concentrations of known cultures of H37Rv, M. fortuitum, M. scrofulaceum and M. avium were used as controls in the study. After 3 weeks of incubation, growth was observed on all the drug free Löwenstein Jensen slants but none of the slants containing PNB, which inhibited the growth of M. tuberculosis. The cultures were further confirmed to be M. tuberculosis by niacin, nitrate and catalase tests. Direct susceptibility to PNB was thus found to be a simple, cheap and technically feasible method of preliminary identification of M. tuberculosis and its effective differentiation from NTM, which may be adapted for use at Level II laboratories, especially in developing countries.