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A method for assaying glucose in serum or plasma samples using a thermostable glucokinase was developed. Glucokinase from Bacillus Stearothermophiluswas coupled with glucose-6-phosphate dehydrogenase to produce NADPH, which reduced the tetrazolium dye MTT to its formazan. Detection of the product at 660 nm allowed samples containing up to 30 mmol/L glucose to be assayed with an endpoint method. Use of the optimal wavelength for formazan detection, 570 nm, increased sensitivity for NADPH detection by over threefold compared to UV detection. The stability of glucokinase assay mixtures was extensively studied, with variation in buffers, salt and enzyme stabilizers. Maximal half life for reagent stability at room temperature was approximately 30 days, with storage of assay mixtures in two solutions. Various drugs and metabolites were tested for interference in the method and no significant interferences were found.