Antibiotics are considered as a cornerstone in the foundations of modern medicine. However, their abuse accelerated the emergence of antibiotics resistant bacteria, resulting in shortened lifespan of antibiotics. In an effort to abolish the antibiotic resistance propagation, I aimed to develop a rapid method for differentiating resistant bacteria in diverse antibiotics treatment using propidium iodide (PI) and alamarblue (AB). PI, a fluorescent nucleic acid-staining dye to detect damaged cell, and AB, a fluorescent dye to detect viable cells with reducing power, were used to determine bacterial viability. Because their availability has not been validated in diverse bacterial genera, prone to become resistant against antibiotics, I aimed to evaluate the applicability of PI and AB to differentiate resistant bacteria from susceptible or intermediate bacteria against antibiotic-treatment of imipenem. Imipenem is a kind of carbapenem antibiotics that have been generally applied to multi-drug resistant (MDR) bacteria. However, recently, carbapenem-resistant MDR pathogens occur world-wide and pose a serious threat to human health. Diagnostic criteria for antibiotic resistance using PI and AB were established using four different pathogenic gram (-) bacteria that frequently acquire antibiotics resistance, including Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. When the optimized diagnostic protocol was tested in 40 clinical isolates, all of them were successfully differentiated with an accuracy of over 90% compared with conventional method. Given that usage of PI and AB provides cost-effectiveness and economy of time, the devised method in this study has the potential for developing a rapid detection kit for antibiotics resistant bacteria.