Myocardial ischemia and the subsequent return of normal blood flow by reperfusion can both lead to significant injury in the heart. Ischemia / reperfusion (I/R) injury is characterized biochemically by an influx of reactive oxygen species (ROS) and calcium overload, which are both thought to induce proteases (e.g. matrix metalloproteinases and calpains) that target contractile proteins and other metabolic processes that are necessary for uninhibited function of the myocytes. Such proteases are therefore assumed to contribute to contractile dysfunction, apoptosis and necrosis during I/R injury. Here we describe a high-throughput approach to identifying and quantifying the targets of proteolysis in I/R.  An ex vivo rat heart model of brief I/R was employed in the presence and absence of ROS scavengers. N-terminal peptides were enriched and quantified by N-terminal amine isotopic labelling of substrates (N-TAILS) and analysed by LC-MS/MS. Degradation products (neo-N-termini) were identified that corresponded to previously identified markers of proteolytic damage in I/R, including the troponins (TnI and TnT) and myosin light chains 1 and 2, as well as completely novel sarcomeric proteins such as desmin and myomesin. Cleaved proteins were also identified in key metabolic pathways including oxidative phosphorylation, the TCA cycle, and redox regulation. Finally, cysteine and glycine rich protein 3 (CSRP3) was confirmed as a target for degradation during I/R, which correlates with our previous work showing the release of CSRP3 peptides into the coronary perfusate post-I/R in both animal models and human STEMI patients.  Collectively, our data confirm the increased activity of proteases during I/R injury and identify new molecular proteolytic targets that may contribute to the functional consequences of I/R in the myocardium.