GHB is an endogenous metabolite in brain and peripheral organs that has many therapeutic uses but is also widely abused for its sedative and memory loss effects. Due to its widespread abuse, there is a substantial need for a reliable and easy assay for GHB that can be used for clinical, abuse, workplace, and law enforcement purposes. Several assays for GHB were developed that utilize the enzyme gamma-hydroxybutyrate dehydrogenase (GHB-DH). GHB-DH from the bacterium Ralstonia eutropha was cloned as a readily isolated and stable fusion protein easily purified by affinity chromatography. The coupling of GHB oxidation to diaphorase-mediated reduction of tetrazolium pro-dye resulted in the formation of a colored product. Michaelis-Menten parameters for oxidation of GHB and ethanol were estimated and the quantitative initial velocity and endpoint versions of the assay in solution are discussed. A semi-quantitative “dipstick” version of the assay on paper is described and is capable of detecting GHB in alcoholic beverages after evaporation of roughly one-fourth drop of beverage. Both assays are sensitive to about 0.05 mg GHB/mL using 10 uL of the sample. Ethanol in urine-level concentrations and agents used to stabilize physiological fluids do not significantly interfere with assay results. The enzymatic assay for GHB is reliable, sensitive, inexpensive, and quick to provide results. The possibilities presented by this screening test warrants further development and validation. Tables, figures, references