Remote ischemic preconditioning (RIPC) induced by brief ischemia/reperfusion cycles of remote organ (e.g., limb) is cardioprotective. The myocardial cellular changes during RIPC responsible for this phenomenon are not currently known. The aim of this work was to identify the activation by phosphorylation of cardiac proteins following RIPC. To achieve our aim we used isobaric tandem mass tagging (TMT) and reverse phase nanoliquid chromatography tandem spectrometry using a Linear Trap Quadropole (LTQ) Orbitrap Velos mass spectrometer. Male C57/Bl6 mice were anesthetized by an intraperitoneal injection of Tribromoethanol. A cuff was placed around the hind limb and inflated at 200 mmHg to prevent blood flow as confirmed by Laser Doppler Flowmetry. RIPC was induced by 4 cycles of 5 min of limb ischemia followed by 5 min of reperfusion. Hearts were extracted for phosphoproteomics. We identified approximately 30 phosphoproteins that were differentially expressed in response to RIPC protocol. The levels of several phosphoproteins in the Z-disk of the sarcomere including phospho-myozenin-2 were significantly higher than control. This study describes and validates a novel approach to monitor the changes in the cardiac phosphoproteome following the cardioprotective intervention of RIPC and prior to index ischemia. The increased level of phosphorylated sarcomeric proteins suggests they may have a role in cardiac signaling during RIPC.