Background <p>Adult murine cardiomyocytes serve as a pivotal model for cardiovascular research. High-quality isolation is essential for studying disease mechanisms and drug screening. Current methods, however, are limited by reliance on specialized equipment and inconsistent efficiency, necessitating a more convenient and reliable protocol.</p> Methods and results <p>We developed an optimized, Langendorff-free method for isolating cardiomyocytes and cardiac fibroblasts from adult mice. Following anesthesia, an enzymatic digestion solution was infused directly via the aortic root, utilizing the coronary circulation to achieve efficient tissue dissociation. This approach yielded adult mouse cardiomyocytes with high viability, intact morphology, and suitability for long-term culture, comparable to the standard Langendorff technique. Functionally, the isolated cardiomyocytes retained robust responsiveness to pathological stimuli, including hypoxia and adrenergic agonists (isoprenaline, norepinephrine, phenylephrine), demonstrated by characteristic alterations in contractile function, relaxation kinetics, and calcium handling. Furthermore, cardiac fibroblasts co-isolated using this protocol could be successfully activated by TGF-β, showing significant upregulation of myofibroblast markers (e.g., α-smooth muscle actin) and pro-fibrotic signaling pathways.</p> Conclusion <p>This study establishes a novel, efficient, and accessible method for the simultaneous isolation of functional cardiomyocytes and cardiac fibroblasts from adult mice. The protocol overcomes key limitations of existing techniques and provides a reliable platform for in vitro mechanistic studies and drug discovery in cardiovascular disease.</p> Graphical Abstract <p></p>