Quorum sensing, the bacterial cell-cell communication by small molecules, controls important processes such as infection and biofilm formation. Therefore, it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-L-homoserine lactones, the most common class of communication molecules among Gram-negative proteobacteria, mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However, identification, characterization, and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here, a (bio)chemical activity assay is described, which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this, the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis to L-homoserine. In turn, L-homoserine is detected by the previously described calcein assay, which is sensitive to ␣-amino acids with free N and C termini. Besides its establishment, the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylaseexpressing Escherichia coli clones in an artificial library. Furthermore, this study indicates that porcine aminoacylase 1 is not active toward N-acyl-L-homoserine lactones as published previously but instead converts the autohydrolysis product N-acyl-Lhomoserine. IMPORTANCEIn this study, a novel method is presented for the identification, characterization, and optimization of quorum-quenching enzymes that are active toward N-acyl-L-homoserine lactones. These are the most common communication molecules among Gram-negative proteobacteria. The activity assay is a highly valuable complement to the available analytical tools in this field. It will facilitate studies on the environmental impact of quorum-quenching enzymes and contribute to the development of therapeutic and technical applications of this promising enzyme class. The bacterial cell-cell communication by small molecules known as quorum sensing (QS) enables these single-celled organisms to coordinate their gene expression among a local population, depending on environmental conditions (1-4). Important processes such as infection and biofilm formation are controlled by QS. Hence, QS is a highly relevant target for naturally occurring and human-made interference termed quorum quenching (QQ), and it is mediated in particular by small-molecule inhibitors and QQ enzymes (5). From an ecological point of view, QQ has a significant influence on the interactions between bacteria (6, 7) and also bacteria and eukaryotes (8). Prospective therapeutic applications of QQ include the prevention a...