Cardiomyocyte maturation represents the final stage of functional development in the heart. During this critical process, fetal cardiomyocytes gradually acquire adult phenotypes and become capable of sustaining billions of contraction–relaxation cycles throughout an organism’s lifespan. This transition is driven by gene expression changes that remodel cardiomyocyte structure, metabolism, and functions. In mice, cardiomyocyte maturation takes place during embryonic day (E)18.5 to postnatal two weeks, during which the heart loses its regenerative capacity in response to injury. Hence, characterizing the functional and molecular changes during mouse cardiomyocyte maturation and how this process is regulated provides invaluable insights into heart development and disease. Cardiomyocyte-specific analysis requires isolating cardiomyocytes from other cardiac cell populations. Here, we present a standardized protocol for consistent isolation of neonatal and postnatal mouse cardiomyocytes. Leveraging enzymatic and mechanical digestion, coupled with non-cardiomyocyte depletion via pre-plating, our protocol enables high-quality cardiomyocyte isolation in approximately three hours. In addition, we detail methods for cardiomyocyte culture, molecular, and functional characterization.