Gram-negative bacteria use outer membrane vesicles (OMVs) for toxin trafficking, immune interference, horizontal gene transfer, antibiotic protection, and cell-cell communication. Despite their direct contribution to many pathogenesis-related behaviors, our understanding of how OMVs are produced remains surprisingly incomplete. The Bilayer Couple model describes the induction of OMV formation resulting from the preferential accumulation of small molecules in the outer leaflet of the membrane, resulting in leaflet expansion and membrane bending. Previous work has highlighted the importance of the structure of the Quinolone Signal (PQS) in driving OMV formation, but the nature of interactions with membrane lipids remains unclear. Our recent in silico analysis suggested that a new interaction, between the PQS ring nitrogen and Lipid A, is critical for PQS function. Here, we used chemical analogs to interrogate the importance of specific PQS functional groups in its ability to stimulate OMV biogenesis. We demonstrated that OMV induction requires the presence of all PQS functional groups together. Further modeling uncovered that PQS prefers interaction with the outer leaflet of the membrane, consistent with its unique ability to drive OMV biogenesis. This was explained by much greater hydrogen bond formation between PQS and Lipid A. Interestingly, the preference of PQS for the outer leaflet coincided with that leaflet becoming crowded. Thus, the initial insertion of PQS into the outer leaflet would be expected to encourage local accumulation of more PQS to drive the induction of membrane curvature and subsequent OMV formation.