Updated project metadata. Ischemia/Reperfusion (I/R) results in altered metabolic and molecular responses. Phosphorylation, a posttranslational modification, is one of the most noted regulatory mechanisms and differential phosphorylation affects numerous regulatory and signaling mechanisms. To further understand the phosphoproteomic changes that occur in the heart during I/R we utilized iTRAQ-based quantitative proteomic and LC-MS/MS techniques to obtain a profile of phosphopeptides expressed under control and I/R conditions. A total of 1896 phosphopeptides were identified, and 116 were assessed as significant. The 116 phosphopeptides represented 87 unique proteins. Analysis of the phosphopeptides using Motif-x identified 2 predominant motifs: arginine and proline-directed serine phosphorylation sites. Two proteins known to be altered by I/R were among the dataset obtained, phospholamban and pyruvate dehydrogenase, and were used to confirm validity of the profile. Functional characterization of the phosphopetides identified in this study could lead to further understanding of the signaling mechanisms involved during I/R damage in the heart, as well as identifying new areas to target therapeutic strategies.