Abstract: Antibiotic resistance persists as serious threat against our ability to treat drug resistant infections. In 2016, teixobactin, a new antimicrobial peptide, was reported to kill Gram-positive pathogens without detectable resistance, a remarkable property that antibiotics lack. Mechanistic studies have shown that teixobactin binds to cell wall precursors, resulting in cellular lysis of bacteria. However, the cellular localization of teixobactin has not been characterized, and doing so would require a fluorescent teixobactin analogue. In my thesis defense, I will show two synthetic methods to construct fluorescent teixobactin probes. The first synthesis yields a rhodamine-labeled analogue, Lys(Rhod)9,Arg10-teixobactin, which retains antibiotic activity and labels the septa and sidewalls of various Gram-positive bacteria. This was first time the cellular localization of teixobactin has been characterized and the Lys(Rhod)9,Arg10-teixobactin probe has applications in studying the mechanism of action of teixobactin, the biosynthesis of peptidoglycan and teichoic acids, and undecaprenyl phosphate metabolism and recycling. The second synthetic method enables labeling of Lys9,Arg10-teixobactin and Lys10-teixobactin with a variety of NHS ester fluorophores. Using the NHS ester labeling strategy, a small library of fluorescent teixobactin analogues was generated, which enabled specialized techniques, such as FRET, to further characterize the mode of action of teixobactin. In the last component of my defense, I will summarize current efforts in using spectral FRET microscopy to determine the presence of quaternary structures of fluorescent teixobactins on live B. subtilis cells. These studies pave the way for further understanding the interesting mode of action of teixobactin and its ability to evade resistance