Increased intestinal or gastric permeability is one of the major hallmarks of liver cirrhosis. The current gold standard for diagnosis of aberrant gut permeability due to disease is the triple-sugar test, where carbohydrates are orally administered and urinary excretion is measured. Hereby, elevated lactulose levels indicate intestinal permeability, whereas increased sucrose levels reveal gastric permeability. However, reliable detection and quantification of these sugars in a complex biological fluid still remains challenging due to interfering substances. Here we used Nuclear Magnetic Resonance (NMR) spectroscopy with a simple and fast protocol, without any additional sample extraction steps, for straight-forward simultaneous quantification of sugars in urine in order to detect increased intestinal and gastric permeability. Collected urine samples were diluted in buffer and one- and two-dimensional proton spectra were recorded in order to reveal carbohydrate concentrations in individual urine samples containing mannitol, sucrose and/or lactulose. Overall, this article presents a fast and robust method for simultaneous quantification of different sugars down to low micro-molar concentrations for research studies and can be further extended for clinical studies with automation of the quantification process.