Protein phosphorylation is one of the most routinely studied post-translational modifications and is involved in a variety of cellular signaling processes. Currently, mass spectrometry is the analysis of choice for detecting global phosphoproteomic changes in biological systems, with the ability to map 100s-1000s of phosphorylation amino acid residues. In the current protocol we describe the enrichment and detection of phosphopeptides from cultured neonatal cardiac myocytes. This in vitro method allows for manipulations of the cellular environment that are impossible to obtain with in vivo models and have been a valued commodity in the unraveling of cardiac signaling pathways.