A semi-automated method that uses the common efflux pump (EP) substrate ethidium bromide (EB) is described for the assessment of EP systems of bacteria. The method employs the Rotor-Gene(TM) 3000 thermocycler (Corbett Research) for the real-time assessment of accumulation and efflux of EB in Phosphate-Buffered Solution (PBS) under varying physiological conditions, such as temperature, pH, presence and absence of the energy source, and presence of efflux pumps inhibitors (EPIs). The method is sufficiently sensitive to characterize intrinsic EP systems of reference strains, a prime necessity if there is a need for assessment of EP-mediated multi-drug resistance (MDR). The method has been successfully applied by us to characterize intrinsic and over-expressed EP systems of Escherichia coli, Salmonella Enteritidis, Enterobacter aerogenes, Enterococcus faecalis and Enterococcus faecium, Staphylococcus aureus, and Mycobacterium smegmatis and Mycobacterium avium, suggesting that if the organism can be maintained in PBS, the system described may suffice for the evaluation and assessment of its EP system.