Through mutation, bacteria have become resistant to current antibiotics; thus, there is a continuous demand for new antibiotics. The compound KKL-896 inhibits the bacterial pathway of trans-translation. To search for the molecular target of KKL-896, experiments (such as: MIC assays, growth curve assays, and P1 transductions) were conducted with E. coli ΔtolC resistant mutants. With knowledge of KKL-896’s target, drug development may advance, and ultimately lead to a new way to combat tuberculosis.