Plant-derived bioactive peptides demonstrate great potential given their availability and cost-effectiveness. In this study, barley (Hordeum vulgare L.) grain proteins were explored for bioactive peptides with potential health applications. Gross grain proteins were obtained through aqueous extraction, after which, trypsin hydrolysis was performed. The hydrolysis process provided high peptide yields reaching 11%. The peptide hydrolysates were evaluated for their antioxidant and metal chelating activities, DPP4 inhibition, deglycation, and antimicrobial activity. The obtained results demonstrated biological activity levels of great importance and comparable to reference molecules. To confirm the experimental results, barley grain protein sequences were simulated for hydrolysis and explored for potential bioactive peptides using the FeptideDB. Indeed, barley grain proteins contain a great diversity of bioactive peptides with various biological activities. The current data highlights the promising therapeutic application of barley-derived bioactive peptides. Further molecular and sequencing approaches are necessary to identify and characterize these peptides.