The depletion of dissolved oxygen in a defined synthetic medium can be measured in real time, using a micro-well plate format, associated with a fluorescent plate reader. This technology is appropriate for investigating the effect of antibiotics on cell kinetics because there is a direct correlation between the latter and the amount of dissolved oxygen in the medium of an assay. In this study, the metabolic activity of the opportunistic human pathogen Pseudomonas aeruginosa PA01 was investigated using the OxoPlate OP96U optical sensor technology. The response of P. aeruginosa to aminoglycoside antibiotics when Ca2+and Mg2+ ions are present in the Evans defined synthetic medium was measured. The results revealed that the effect of antibiotics on P. aeruginosa is influenced by the concentration of divalent cations present in the test medium, although the efficiency of Ca2+ in supressing antibiotic activity was found to be greater than that of Mg2+. By comparison to tobramycin, the effect of amikacin is largely inhibited by the Ca2+and Mg2+concentrations. The study results underscore that the reliability of the observation of growth inhibitors is enhanced by the oxygen consumption measurements. Thus, the OxoPlate OP96U system is proven to be an accurate method to test the effectiveness of antibiotic treatments against P. aeruginosa.