A direct assay for chloramphenicol acetyl transferase (CAT) has been presented wherein the assay reagent comprises chloramphenicol, an acyl CoA compound, and a tetrazolium salt, and wherein the reagent does not have any added coupling redox enzymes. In one embodiment, the reagent is mixed with the test sample and the presence of CAT is detected by an optical response. In a second embodiment, the reagent is mixed with a test sample containing CAT and the optical response is quantitated by comparison with standards to measure CAT activity in the test sample. In another embodiment, an exogenous electron carrier such as phenazines may be used to enhance the detection of CAT. The assay for the presence or activity of CAT can be used in a high-throughput screening assay or to detect CAT as a reporter gene for measuring the expression of a gene of interest. Kits containing the reagents are also provided.