Pseudomonas aeruginosa cells communicate with each other via the secretion and sensing of quorum sensing (QS) molecules, which collectively control the expression of multiple virulence factors. One of these QS molecules is the Pseudomonas Quinolone Signal (PQS), which is biosynthesized in the cytoplasm. Due to the hydrophobic nature of PQS, it is not freely diffusible across the aqueous periplasm separating the inner and outer membranes. However, the mechanisms by which PQS is transported across the cell envelope during secretion and uptake are not known. Here, we exploited metabolic labelling to introduce alkyne and diazirine functionalities into PQS during biosynthesis, and employed these bifunctional analogs for the profiling of PQS-binding proteins. We identified several membrane proteins that bind PQS, and showed that an ABC transporter and two RND efflux systems are functionally important for the uptake and secretion processes of PQS, respectively. Our work provides fundamental insights into PQS-mediated quorum sensing, and reveals potential targets for the development of novel anti-virulence agents.