AbstractQuorum sensing is cell‐to‐cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N‐acyl‐l‐homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram‐negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well‐documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC‐based, C−S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl‐CoA‐ and acyl‐ACP‐utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds.