Metabolic engineering stands to transform the discovery and production of a wide range of chemicals, but metabolic engineering currently demands considerable resource investments that restrict commercial application. To facilitate the applicability of metabolic engineering, general high-throughput and readily implemented technologies are needed to assay vast libraries of strains producing desirable chemicals. Toward this end, we describe here the development of a yeast three hybrid (Y3H) assay as a general, high-throughput, versatile and readily implemented approach for the detection of target molecule biosynthesis. Our system detects target molecule biosynthesis through a change in reporter gene transcription that results from the binding of the target molecule to a modular protein receptor. We demonstrate the use of the Y3H assay for detecting the biosynthesis of tetracyclines, a major class of antibiotics, based on the interaction between tetracyclines and the tetracycline repressor protein (TetR). Various tetracycline derivatives can be detected using our assay, whose versatility enables its use both as a screen and a selection to match the needs and instrumentation of a wide range of end users. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating between producer and nonproducer strains of the natural product tetracycline TAN-1612. The Y3H assay is superior to state-of-the-art HPLC-MS methods in throughput and limit of detection of tetracycline derivatives. Finally, our establishment of the Y3H assay for detecting the biosynthesis of a tetracycline supports the generality of the Y3H assay for detecting the biosynthesis of many other target molecules.