While natural products constitute an established source of lead compounds, the classical iterative bioassay-guided isolation process is both time- and labor-intensive and prone to failing to identify active minor constituents. (HP)TLC.bioautography.MS/NMR, which combines cutting-­edge microbiological, chromatographic, and spectrometric technologies, was developed 10 accelerate anti-tuberculosis (TB) drug discovery from natural sources by acquiring structural information at a very early stage of the isolation process, Using the avirulent, bioluminescent Mtb strain mc²7000 luxABCDE, three variations of bioautography were evaluated and optimized for sensitivity in detecting anti-T``B agent , including established clinical agents and new lead with novel mechanisms of action. Several exemplary applications of this approach to microbial extracts demonstrate its potential as a routine method in anti-TB drug discovery from natural sources.