To develop a probe-based triplex quantitative real-time PCR assay to simultaneously detect the upregulation of the efflux pumps AmrAB-OprA, BpeAB-OprB and BpeEF-OprC in Burkholderia pseudomallei strains exhibiting increased minimum inhibitory concentrations toward meropenem, doxycycline or trimethoprim-sulfamethoxazole. The triplex assay was developed and subsequently tested on RNA isolated from eight clinical and eight laboratory-generated B. pseudomallei mutants harboring efflux pump regulator mutations. The triplex assay accurately detected efflux pump upregulation in all clinical and laboratory mutants, which corresponded with decreased antibiotic susceptibility or antibiotic resistance. Rapid detection of antibiotic resistance provides clinicians with a tool to identify potential treatment failure in near real time, enabling informed alteration of treatment during an infection and improved patient outcomes.