Fatty acids are essential building blocks used in the assembly of many important bacterial components such as phospholipids, lipoproteins and lipopolysaccharides. Because of this type II fatty acid biosynthesis (FASII) has become an attractive target for mechanism-based drug discovery approaches1-5. In the search for FASII inhibitors, both enzymatic and whole cell based screens have been used. Unlike in vitro enzymatic assay screens, whole cell based assays have the advantage of only selecting compounds that are able to penetrate cells and reach intracellular targets. Despite this advantage, most of the compounds found in whole cell screens show poor target selectivity. Here we describe a target selective S. aureus whole cell assay that combines agar-diffusion and protein over expression techniques. This agar based two-plate differential sensitivity assay was used to help confirm the newly discovered antibiotic platensimycin inhibited bacterial growth by specifically targeting the essential FASII enzyme FabF6.