Polymyxins are considered as the last-resort antibiotics for the treatment of bacterial infections, however, the prompting concern of polymyxin-resistant bacteria demands faster antimicrobial susceptibility testing method to guide reasonably therapy in clinic. Traditional antimicrobial susceptibility testing methods are usually time-consuming. Here, we have designed and synthesized a fluorescent polymyxin-fluorophore derivative, Polymyxin-NBD, which has been successfully applied to develop a flow cytometry assay that can rapidly determine polymyxin B susceptibility of various Gram-negative species including Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The short time frame, easy data analysis, and application to multiple species for this qualitative test make it a promising antimicrobial susceptibility test method.