Whole-cell biosensors, based on the visualization of a reporter strain’s response to a particular stimulus, are a robust and cost-effective means to monitor defined environmental conditions or the presence of chemical compounds. One specific field in which such biosensors are frequently applied is drug discovery, that is, the screening of large numbers of bacterial or fungal strains for the production of antimicrobial compounds. Here, we describe the application of a luminescence-based Bacillus subtilis biosensor for the discovery of cell wall active substances; this article is an update to our previous chapter published in 2017. The system is based on the well-characterized promoter P_liaI, which is induced in response to a wide range of conditions that cause cell envelope stress, particularly antibiotics that interfere with the membrane-anchored steps of cell wall biosynthesis. A simple “spot-on-lawn” assay, where colonies of potential producer strains are grown directly on a lawn of the reporter strain, allows for quantitative and time-resolved detection of antimicrobial compounds. Due to the very low technical demands of this procedure, we expect it to be easily applicable to a large variety of candidate producer strains and growth conditions.