An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureus agr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC 50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I–III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I–III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus . The research strategy here reported can be extended to the rest of the AIP types of S. aureus , allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients.