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The ability to produce new molecules of potential pharmaceutical relevance via combinatorial biosynthesis hinges on improving our understanding of acyl-carrier protein (ACP)-protein interactions. However, the weak and transient nature of these interactions makes them difficult to study using traditional spectroscopic approaches. Herein we report that converting the terminal thiol of the E. coli ACP 4′-phosphopantetheine arm into a mixed disulfide with 2-nitro-5-thiobenzoate ion (TNB−) activates this site to form a selective covalent cross-link with the active site cysteine of a cognate ketoacyl synthase (KS). The concomitant release of TNB2−, which absorbs at 412 nm, provides a visual and quantitative measure of mechanistically relevant ACP-KS interactions. The colorimetric assay can propel the engineering of biosynthetic routes to novel chemical diversity by providing a high-throughput screen for functional hybrid ACP-KS partnerships as well as the discovery of novel antimicrobial agents by enabling the rapid identification of small molecule inhibitors of ACP-KS interactions.