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Acetyl–coenzyme A (CoA) carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacteria and eukaryota. This enzyme is the target of drug design for treatment of human metabolic diseases and of herbicides acting specifically on the eukaryotic form of the enzyme in grasses. Acetyl–CoA carboxylase activity screening in drug and herbicide design depends mostly on a time-consuming enzyme assay that is based on the incorporation of radiolabeled bicarbonate into the product malonyl–CoA. Here we describe a new simple, continuous, and quick photometric assay avoiding radioactive substrate. It couples the carboxylation of acetyl–CoA to the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of malonyl–CoA, which is catalyzed by recombinant malonyl–CoA reductase of Chloroflexus aurantiacus. This assay can be adapted for high-throughput screening.