Measuring cardiomyocyte nuclear ploidy is crucial for evaluating broader aspects of cardiac development, function, and disease progression. Fluorescence in situ hybridization (FISH) remains the gold standard for ploidy identification
however, its application in cardiomyocytes is hindered by their unique cellular complexities. Here, we describe a detailed cardiomyocyte-specific FISH (cardioFISH) protocol. CardioFISH incorporates a tailored enzymatic digestion strategy to enhances nuclear accessibility while preserving cellular integrity and minimizing sarcomere-derived autofluorescence. Additionally, we introduce a 3D nuclear visualization framework for comprehensive cardioFISH signal analysis, addressing the limitations imposed by the large nuclear dimensions of cardiomyocytes, where signals are frequently distributed across multiple imaging planes. This two-day cardioFISH protocol is applicable to various stages of cardiomyocyte development and provides a powerful tool for advancing studies of cardiomyocyte ploidy.