Purified bovine β-casein was digested with glutamyl endopeptidase (GE) at 37 and 50 °C for 4 h. The peptides generated were determined using nano-LC-ESI-qTOF-MS/MS. GE was highly specific and hydrolyzed peptide bonds in β-casein predominantly on the carboxy terminal of Glu and Asp. Pro residues were not preferred, while Met was poorly preferred at the P1' position. Glu-Met hydrolysis was less preferred in comparison to Asp-Met hydrolysis. Five synthetic peptides corresponding to specific sequences in β-casein were incubated with GE at 37 °C to further characterize the substrate specificity. MS analysis of the digestion products indicated that GE hydrolyzed Glu-Ser in Glu-Glu-Ser. Furthermore, hydrolysis of Glu-Met and Glu-Pro was observed. The presence of multiple-phosphorylated Ser residues upstream from the scissile bond did not appear to affect hydrolysis of Glu-Ser. The results herein are relevant to our understanding of the substrate specificity of GE and the peptides that may be expected during the hydrolysis of β-casein.