Neonatal rodent cardiomyocytes serve as a valuable in vitro model for investigating the cellular and molecular mechanisms of the heart, offering an alternative to the technical challenges associated with isolating and maintaining adult cardiomyocytes. Importantly, studying atrial and ventricular cardiomyocytes separately is crucial due to their distinct functional and genetic properties. Here, we present a protocol for the simultaneous isolation and culture of neonatal mouse atrial and ventricular cardiomyocytes. This procedure includes the dissection of neonatal mouse hearts at postnatal day 0 to day 2(P0–P2), separation of atrial appendages and ventricles, and sequential enzymatic digestion using trypsin and collagenase. The chamber-specific identities of isolated cardiomyocytes were validated through gene expression analyses and immunofluorescence staining with atrial and ventricular markers. The resulting cardiomyocyte populations exhibit high viability and retain chamber-specific characteristics, rendering them suitable for a wide array of experimental applications under both physiological and pathological conditions. Potential applications include physiological assessments (e.g., calcium transient analysis and patch clamp), morphological studies, biochemical assays, and drug screening.