The frequency of polymyxin-resistant pathogenic Gram-negative bacteria appearing in the clinic is increasing, and the consequences are largely mediated by modification of lipopolysaccharide (LPS) in the outer membrane. As polymyxins exert their antibacterial effect by binding to LPS, understanding their mode of binding will prove highly valuable for new antibiotic discovery. In this study, we assess the potential of MIPS-9451, a fluorescent polymyxin analogue designed for imaging studies, as a fluorescent reporter molecule, titrating it against 17 different Gram-negative species and/or strains of LPS. MIPS-9451 bound to the various species and/or strains of LPS with a dissociation constant ranging between 0.14 ± 0.01 µM ( Escherichia coli) and 0.90 ± 0.42 µM ( Porphyromonas gingivalis) (mean ± SE). Furthermore, we assessed the applicability of MIPS-9451 to assess affinities of polymyxin B to different LPS species in a displacement assay which yielded inhibition constants of 6.2 µM ± 33%, 7.2 µM ± 30% and 0.95 µM ± 13% for Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella enterica, respectively (mean ± CV). The results from this study are concordant with those observed with similarly structured polymyxin probes, confirming the potential for MIPS-9451 for quantitation of polymyxin-LPS affinities in discovery programs of novel polymyxin antibiotics.