Bacterial quorum sensing (QS) is being contemplated as a promising target for developing innovative diagnostic and therapeutic strategies. Here we report for the first time the development of antibodies against 2-heptyl-4-quinolone (HHQ), a signaling molecule from the QS system of , involved in the production of important virulent factors and biofilm formation. The antibodies produced were used to develop an immunochemical diagnostic approach to assess the potential of this molecule as a biomarker of infection. The ELISA developed is able to reach a detectability in the low nM range (IC = 4.59 ± 0.29 nM and LOD = 0.34 ± 0.13 nM), even in complex biological samples such as Müeller Hinton (MH) culture media. The ELISA developed is robust and reproducible and has been found to be specific to HHQ, with little interference from other related alkylquinolones from the QS system. The ELISA has been used to analyze the HHQ production kinetics of clinical isolates grown in MH media, pointing to its potential as a biomarker of infection and at the possibility to use the technology developed to obtain additional information about the disease stage.