2-alkyl-4-quinolones (AQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) are quorum sensing signal molecules. Here we describe two methods for AQ detection and quantification that employ thin layer chromatography (TLC) and microtitre plate assays in combination with a lux-based Pseudomonas aeruginosa AQ biosensor strain. For TLC detection, organic solvent extracts of bacterial cells or spent culture supernatants are chromatographed on TLC plates, which are then dried and overlaid with the AQ biosensor. After detection by the bioreporter, AQs appear as both luminescent and green (pyocyanin) spots. For the microtitre assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AQ biosensor. Light output by the bioreporter is proportional to the AQ content of the sample. The assays described are simple to perform, do not require sophisticated instrumentation, and are highly amenable to screening large numbers of bacterial samples.