One of the challenges in antibiotic lead discovery is the difficulty and time-consuming task of determining the mechanism of action (MOA) of antibacterial compounds. In this report, we describe the development and validation of a facile and inexpensive assay system utilizing disk diffusion of inhibitors on solid agar medium embedded with mixed pools of a comprehensive collection of Escherichia coli clones each containing a plasmid-borne inducible essential gene from E. coli. From individual clones, pilot small-scale (48 or 50 clones) assays, to full-scale target identification platform for antibacterials (TIPA) system, involving a variety of assay formats (liquid vs solid media, individual vs mix clones), we demonstrate that elevated resistance phenotypes of relevant cell clones were highly specific. In particular, the TIPA system was able to reveal cellular targets of several known antibacterial inhibitors: cerulenin, diazaborine, indolmycin, phosphomycin, and triclosan. Complementary to several existing MOA profiling schemes, the TIPA system offers a simple and low-cost method for elucidating the target proteins of antibacterial inhibitors, thus will facilitate discovery and development of novel antibacterial compounds to combat multidrug-resistant bacterial pathogens.