The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and α-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.