5-Hydroxymethylfurfural (5-HMF) is a natural occurring substance taken up by everyday food. In former studies it was shown that 5-HMF is completely decomposed in the body after oral or intravenous application resulting in three main metabolites named 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine, and possibly a forth metabolic substance, termed 5-sulphoxymethylfurfural, is formed. Determination is possible via HPLC using a hydrophilic interaction chromatography (HILIC) column with an appropriate gradient system (ACN/ammonium formate 100 mM, pH 2.35). Urine samples were purified by use of an SPE method beforehand working with ScreenA cartridges. This cleaning procedure was validated based on ICH guidelines in terms of linearity, quantification, and detection limit, as well as precision, repeatability, and accuracy. Analysis of real-life samples coming from two healthy probands and one cancer patient, who all received 240 mg 5-hydroxymethylfurfural orally once a day, showed dicarboxylic acid and the glycine conjugate in their urine samples. Recovery of the initial compound in form of transformed metabolites was up to 90% within 48 h. Potentially toxic 5-sulphoxymethylfurfural could not be found.