Isolated single cardiomyocytes are commonly used to understand fundamental cardiac cell physiology and cell signaling at the molecular level. The protocol to isolate cardiomyocytes is under continual optimization between laboratories, despite being conducted for many years. The delicate nature of these cells coinciding with laboratory equipment variations can make the consistency of isolation challenging. The protocol described here presents a simple method to routinely isolate high-quality cardiomyocytes using Langendorff perfusion. This method provides cells well suited for analysis of physiological cellular calcium handling and spontaneous calcium release.