Considering the increased concerns with controlling infectious epidemics such as tuberculosis, a global concerted effort (WHO) is now dead-lined to tackle the emergence of extensive drug resistance through identifying a novel line of therapeutics which will on the one hand shorten the course of treatment and on the other is also expected to be effective against the emerging resistant strains. Major problems with the preclinical drug screening against the uniquely slow-growing pathogen Mycobacterium tuberculosis are either found expensive, time-consuming, or require a highly complex laboratory setup. A rapid and convenient, although relatively inexpensive, method requiring very little consumption of inhibitors within a simple microbiology setup for antimycobacterial screening is thus timely. The spot-culture growth inhibition assay aims to test the biological activity of a number of newly discovered natural products and thousands of novel chemicals synthesized on the basis of basic structural and molecular biology studies. Many different classes of novel chemical entities are now independently prepared around the world by distinguished chemists on the chemical behavior of the group of molecules. To serve the purpose of antimycobacterials screening, we aim to describe a method in this chapter performed in a six-well plate format. This method can also be extended accurately to a 96-well plate format according to the necessity of the project. In addition to evaluating a range of prospective drug candidates, this method would also contribute to elucidate substrates for many putative endogenous pathways through comparing the chemical inhibition with the corresponding genetic modification.