Post-translational modifications to myofilament proteins are essential for the regulation of cardiac function in both normal and disease states. Recent developments in the field of proteomics have produced a variety of useful tools to study protein modifications. Current applications of proteomic technologies in the study of modifications to myofilament proteins are summarized. The separation, identification and characterization of myofilament modifications using gel electrophoresis and mass spectrometry approaches are discussed. Each method is illustrated and evaluated with selected examples, and several powerful emerging technologies are assessed.