A liquid chromatography with tandem mass spectrometry method was developed for the determination of 27 glutamyl di- and tripeptides in food ingredients. Such compounds are of importance for the food industry, as they can modulate the perception of basic tastes (sweet, salty, and umami). Due to their high polarity, the hydrophilic interaction chromatography mode was selected to have sufficient retention on the column and the best separationwas obtained on an amide hybrid silica stationary phase packed with 1.7 μm particles. Thorough optimization of the mobile phase was performed as the start-composition had to be free of ammonium to avoid on-column cis–trans isomerization of the first eluting proline dipeptide.Abaseline separationwas achieved for all α and γ isomers whereas only a partial resolution was obtained for γ-Glu-Leu and γ-Glu-Ile, for which only the position of a methyl group differs. A fast sample preparation,based on successive dilutions, was performed before injection into the liquid chromatography with tandem mass spectrometry system. The developed method was then applied for the semi-quantification of glutamyl di- and tri-peptides in four different food ingredients. The methodology will further support the ptimization of production processes to select the conditions for which the peptide concentrations would be the highest.