Lactulose is becoming the disaccharide of choice in the dual sugar assessment of passive permeability of the small intestinal mucosa. However its more widespread use is hampered by current analytical methods which are tedious and time consuming. An automated spectrophotometric technique for the assay of this sugar in urine is presented in which lactulose is linked by a series of enzyme reactions to the equimolar production of NADPH. In addition to lactulose, the procedure also gives accurate values for lactose, glucose and fructose in the urine sample. The assay has been shown to be highly specific for lactulose and lactose and was not affected by high concentrations of other sugars or other urinary constituents. Within assay and between assay precision were similar with the coefficient of variation for both sugars in the range 0.4-1.6%. The technique represents a significant improvement in time, simplicity and precision on existing methods of analysis.