Barley (Hordeum vulgare L.
Poaceae), the second most important grain after wheat, contains phenolamides, specifically hordatines and their agmatinated precursors. Hordatines are the unique compounds found in barley, consumption of which is associated with beneficial effects for human health. This study investigated the impact of germination on the concentrations of barley phenolamides by analyzing their kinetic changes in whole barley seedlings and their distribution across shoots, seeds, and roots over a 9-day germination period under light and dark conditions. To obtain authentic standards, hordatines A and C (HA and HC) were synthesized, while hordatine B (HB) and glycosylated hordatines A and B (HAG and HBG) were isolated from a standardized hordatine-rich fraction (HRF) developed using ion exchange resins. The chemical structures were established using