Cardiovascular diseases are one of the leading causes of death worldwide with ischemic heart disease the largest contributor. To minimise ischemic damage, timely reperfusion is essential to salvage the affected tissue however, reperfusion itself can result in further damage. While a number of key signalling pathways are known to be involved in reperfusion injury, the key activators and temporal profile of such signalling proteins is unknown. The current study utilises large scale phosphoproteomics to monitor the activation/repression of signalling pathways during the reperfusion period. Rat hearts were subjected to 15 minutes of global ischemia (15I) by Langendorff perfusion with periods of reperfusion spanning 1-, 2-, 5-, 15- and 60-minutes. Peptides were labelled with TMT prior to enrichment of phosphopeptides utilising the TiSH method, with analysis by HCD tandem MS. This study identified 18,760 phosphopeptides originating from 4,915 proteins. Using a z-score cutoff of ±1 we observed 8,025 phosphopeptides significantly regulated over the timecourse of 60 minutes reperfusion, with 5,189 of these occurring within the first 2 minutes of reperfusion. These regulated phosphopeptides were mapped to functional pathways utilising STRING and KEGG to a number of signalling pathways including MAPK, cGMP-PKG and PI3K-Akt signalling as well as a number of pathways responsible for contractility and cellular structure including the actin cytoskeleton, calcium signalling, tight junction and focal adhesion. By elucidating potential signalling proteins responsible for the initiation of reperfusion injury, novel therapeutic targets are possible to improve cardiovascular outcome after an ischemic event, such as the focal adhesion kinase which shows >2 fold increase in phosphorylation indicative of increased activity and is a key activator of a number of signalling pathways which can result in apoptosis.