Barley (Hordeum vulgare) is mostly used in feed and malt production but has the ability to provide humans nutritional benefits. The current wheat based “barley” breads can unfortunately not exceed more than 20% barley flour mixed into the dough due to poor leavening properties. Therefore the objective is to develop barley varieties with good baking properties. The poor leavening properties of barley can be attributed, at least partially, to the physical properties of the storage proteins. Studies based on small scaled baking trials and protein pattern analysed by SDS-PAGE on a large number of high protein barley cultivars suggested differences in baking quality and correlation between baking quality and D-hordeins. The amino acid composition was measures using the newly developed AccQ Tag Ultra Amino acid (AA) derivatisation system designed for the Acquity UPLC. Obtained results from the total AA composition and the hordein pattern indicated that there are genetic variations not only in the distributions of the hordein polypeptides but also in the relative proportions of the storage proteins affecting the AA compositions. The free AA composition in the grain may also provide us the opportunity to give a forecast of the taste of the bread, as the AA composition is known to control certain aspects of the taste. We uses a MSE approach on a time of flight instrument coupled to a UPLC and in gel digestion to identify and characterize the different D-hordeins responsible for baking quality. Digesting the proteins with chymotrypsin results in approx 60% sequence coverage in Biopharmalynx but higher sequence coverage is expected using both chymotrypsin and trypsin as overlapping peptides are produced in the optimal mass range. Sequencing the digested peptides also revealed post translational modifications of the identified hordein which needs further investigation. Our results strongly indicate genetic variations in the relative proportions of the storage proteins affecting the amino acid compositions and baking quality.