This two-step enzymatic reaction uses glucose oxidase, peroxidase and a chromogen. Glucose oxidase catalyze the formation of gluconic acid and hydrogen peroxide via the oxidation of glucose. Peroxidase catalyzes the reaction of hydrogen peroxide with a chromogen via the oxidation of chromogen to colors ranging between green to gray-blue.

Glycosuria may occur in situations, such as pregnancy, in which the increased filtered load may exceed renal tubular reabsorption capacity. Rarely corticosteroid therapy may cause a combination of mild hyperglycemia and increased renal glomerular filtration, producing glycosuria. Renal glycosuria due to a low renal threshold for glucose occurs either as an isolated phenomenon or as part of tubular damage. Because these causes of glucosuria are rare and glucosuria usually indicates significant hyperglycemia, a positive screening test for urine glucose is significant and indicates a substantial likelihood of diabetes mellitus in the nonpregnant patient.