Since the introduction of penicillin into the clinic in 1942, antibiotics have saved the lives of millions of people around the world. While penicillin and other traditional broad spectrum antibiotics were effective as monotherapies, the inexorable spread of antibiotic resistance has made alternative therapeutic approaches necessary. Compound combinations are increasingly seen as attractive options. Such combinations may include: lethal compounds; synthetically lethal compounds; or administering a lethal compound with a nonlethal compound that targets a virulence factor or a resistance factor. Regardless of the therapeutic strategy, high throughput screening is a key approach to discover potential leads. Unfortunately, the discovery of biologically active compounds that inhibit a desired pathway can be a very slow process, and an inordinate amount of time is often spent following up on compounds that do not have the desired biological activity. Here we describe a pathway-directed high throughput screening paradigm that combines the advantages of target-based and whole cell screens while minimizing the disadvantages. By exploiting this paradigm, it is possible to rapidly identify biologically active compounds that inhibit a pathway of interest. We describe some previous successful applications of this paradigm and report the discovery of a new class of d-alanylation inhibitors that may be useful as components of compound combinations to treat methicillin-resistant Staphylococcus aureus (MRSA).