A kinetic assay method based on bacterial bioluminescence and the glycerol dehydrogenase (GDH) enzyme reaction has been developed for the determination of glycerol. The assay system involves the use of three coupled enzyme reactions in which the participating reactants are optimized to allow internal calibration by known amounts of glycerol. This bioluminescent assay method is also suitable for measuring GDH enzyme activity. The lower detection limit for glycerol is 500 pmol and for GDH, 0.001 mU, the assay being linear up to 300 nmol of glycerol and 3 mU of GDH. The percentage recovery of glycerol from serum was 95-100%. This assay method is rapid, sensitive, and reproducible.