Abstract <p>A need exists for a rapid and straightforward method to measure antibiotic susceptibility in bacteria to optimize antibiotic usage and mitigate the emergence of antimicrobial resistance. In this study, we investigated a novel, facile, and rapid colorimetric method for assessing bacterial antibiotic susceptibility via the extracellular electron transfer (EET) assay. This method relies on the colorimetric detection of EET from bacterial respiratory metabolism to an EET reagent containing an electron transfer mediator (riboflavin) and a detectable electron acceptor (methylene blue). The minimum inhibitory concentration (MIC) values obtained for three real clinical nosocomial pathogens <i>(Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae</i>) using our technique align closely with those derived from the gold standard micro-dilution broth assay. Employed riboflavin as an external mediator for electron transfer, enables to screen antibiotic susceptibility of bacterial strains with different capabilities of EET. Our innovative approach offers a rapid assay for identifying EET, and strain-specific effectiveness of antibiotics with diverse mechanisms of action. As we employed simple and cost-effective equipment, there is potential for this method to evolve into a practical approach for simplified antibiotic susceptibility testing.</p>