Seed dormancy is important trait for plant to grow in favorable condition. It is strongly maintained in freshly harvested seed, but impaired in after-ripened seeds. In dormancy regulation, phytohormone abscisic acid (ABA) has essential role. It is transduced thorough protein phosphorylation and occur induction and maintenance of dormancy. To understand the role of ABA during after-ripening, ABA responsive phosphoproteins should be understood. Phosphoproteomic analysis was carried out by using following process. Isolated barley seed embryo was used for protein extraction. Trypsin digestion and phosphopeptide enrichment by HAMMOC method were performed, then purified peptide sample was analyzed by TripleTOF5600 (AB-SCIEX).