Umami is one of the five basic tastes beside sweet, bitter, sour, and salty. It is well known in various fermented food preparations like soy sauce. Umami represents the taste of L-glutamate and 5’-ribonucleotides, such as guanosine and inosine monophosphate. Since the acceptance of the consumers for monosodium glutamate (MSG) is steadily declining, the interest of the food industry for umami active substitutes increased continuously. This study was based on the umami taste of wheat gluten hydrolysates. Differently produced hydrolysates were examined. At the beginning of this project, the overall umami taste of the samples and the peptide composition were determined. Up to 197 small biomolecules were identified, among them several umami active compounds like Ile-Glu, Val-Glu, Val-Asp, Ser-Glu, Glu-Gln-Glu, Val-Val, pGlu-Pro, pGlu-Gln, and pGlu-Gly. The composition of wheat gluten hydrolysate has not previously been described in such detail. A fractionation approach was performed to generate samples containing a lower number of substances while still imparting the umami taste.Fractionation via Size Exclusion Chromatography (SEC) led to three fractions eliciting intense umami taste. A fivefold increase of the umami taste was described by 93 % of the panellists. As before, their peptide composition was determined by UPLC-HR-QTOF-MS/MS. Several peptides were identified, which made the sub-fractions unique, and the known umami active compounds of the starting material were found again. However, the multitude of identified substances made it impossible to discover a single substance that imparted the umami taste. Another fractionation was performed. Sub-fractionation via prepHPLC did not lead to samples showing a significant umami taste in the sensory analysis, even though known umami active substances were identified (Glu-Leu, Val-Glu, Val-Gly, Val-Asp, Pro-Glu, Pro-Gly, Pro-Thr, and diketo-Glu-Gln). A refined SEC sub-fractionation approach led to taste active sub-fractions. Only 17 substances were identified in the sub-fractions, whereas two (Glu-Leu, diketo-Glu-Pro) of them had known umami activities. Sensory analysis of the remaining 15 single compounds needs to be performed to discover a compound with umami activity, which was not described yet.