Abstract Pathogenic mycobacteria are generally slow growing organisms and it takes several weeks to evaluate inhibitors of growth. Therefore, for rapid screening of the inhibitors of mycobacterial growth, a β-galactosidase reporter system has been described which utilises a recombinant Mycobacterium smegmatismc 2155 expressing E. coli lacZgene as the test organism. The assay is based on production of β-galactosidase in presence of drugs during growth. A correlation between β-galactosidase production and colony forming ability of mycobacteria was obtained. β-galactosidase production was inhibited within 6 h by front line standard antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, pyrazinamide and ofloxacin at their reported MICs. The assay was performed on mycobacterial cells permeabilized with chloroform and sodium dodecyl sulfate.