The micro interface between two electrolyte solutions (micro-ITIES) has been exploited as a miniaturized electrochemical platform for detection of Pseudomonas aeruginosa. Electrochemical detection methods for point-of-care-devices in healthcare environments are ideal owing to their small equipment footprint and relative ease of use, while simultaneously, disposable electrode materials are easily fabricated. Meanwhile, P. aeruginosa is a pathogenic bacteria that poses a serious health risk to patients with compromised immune systems. Herein, a proof-of-concept electrochemical method is demonstrated that employs a micro-ITIES between water and oil (w/o) held at the tip of a glass capillary which targets two quorum sensing (QS) molecules produced throughout the bacteria’s life-cycle: 4‐hydroxy‐2‐heptylquinoline (HHQ) and 2‐heptyl‐3,4‐dihydroxyquinoline (PQS, Pseudomonas quinolone signal). QS molecules are used to signal colony growth in a pseudo-multicellular fashion; however, HHQ and PQS are also known virulence factors and are unique to this form of pathogenic bacteria. HHQ and PQS show excellent solubility in chlorinated organic solvent with limited partitioning to the aqueous phase. Moreover, as observed electrochemically, they facilitate proton transfer across the w/o interface which can be monitored/quantified and is exploited here as a means for early bacterial detection. This work demonstrates that the micro-ITIES can serve as a viable platform for P. aeruginosa identification and quantification. Density functional theory calculations were also performed to determine the proton affinities and gas-phase basicities of HHQ/PQS, as well as elucidate the likely site of stepwise protonation within each molecule.