Objective: The purpose of this study was to develop a gas-liquid chromatography method for the determination of the urinary excretion of two nonmetabolizable sugars (lactulose and mannitol) to estimate the intestinal permeability. Methods: Two internal standards (alpha-methyl-D-glucopyranosyde and sucrose) were added to the urine samples prior to derivatization with the aid of a solution of pyridine, [N, O-bis (trimethylsilyl)]-acetamide and chlorotrimethylsilan. Sample preparation was simpler and faster than in other previous methods acid the four sugars were resolved within 27 min. Results: The inclusion of internal standards reduced the coefficient of variation from 9.44% to 4.78%. The method provided better sensitivity, range of analysis, and linearity than a previously published HPLC method reducing variances in the recovery of lactulose and mannitol added to urine, The clinical application of this method was tested in preterm infants fed human milk or cow’s milk based formula and in breast fed term infants. Conclusion: The method described here for the determination of urinary lactulose and mannitol is more rapid, simpler, and more accurate than other previously published methods.