Quorum sensing (QS) is a bacterial cell density-based communication system using low molecular weight signals called autoinducers (AIs). Identification and quantification of these molecules could provide valuable information related to the stage of colonization or infection as well as the stage of the disease. With this scenario, we report here for the first time the development of antibodies against the PQS (pseudomonas quinolone signal), the main signaling molecule from the pqs QS system of Pseudomonas aeruginosa, and the development of a microplate-based enzyme-linked immunosorbent assay (ELISA) able of quantifying this molecule in complex biological media in the low nanometer range (LOD, 0.36 ± 0.14 nM in culture broth media). Moreover, the PQS ELISA here reported has been found to be robust and reliable, providing accurate results in culture media. The technique allowed us to follow up the PQS profile of the release of bacterial clinical isolates obtained from patients of different disease status. A clear correlation was found between the PQS immunoreactivity equivalents and the chronic or acute infection conditions, which supports the reported differences on virulence and behavior of these bacterial strains due to their adaptation capability to the host environment. The results obtained point to the potential of the PQS as a biomarker of infection and to the value of the antibodies and the technology developed for improving diagnosis and management of P. aeruginosa infections based on the precise identification of the pathogen, appropriate stratification of the patients according to their disease status, and knowledge of the disease progression.