Cardiac regeneration in newborn rodents depends on the ability of pre-existing cardiomyocytes to proliferate and divide. This capacity is lost within the first week of postnatal development when these cells rapidly switch from hyperplasia to hypertrophy, withdraw from the cell cycle, become binucleated, and increase in size. How these dynamic changes in size and ploidy impact cardiomyocyte proliferative potential is not well understood. In this study, we innovate the application of a commercially available digital holographic imaging microscope, the Holomonitor M4, to evaluate the proliferative responses of mononucleated diploid and binucleated tetraploid cardiomyocytes. This instrument coupled with the powerful Holomonitor App Suite software enables long-term label-free quantitative three-dimensional tracking of primary cardiomyocyte dynamics in real-time with single-cell resolution. Our digital holographic imaging results provide direct evidence that mononucleated cardiomyocytes retain significant proliferative potential as most can successfully divide with high frequency. In contrast, binucleated cardiomyocytes exhibit a blunted response to a proliferative stimulus with the majority not attempting to divide at all. Nevertheless, some binucleated cardiomyocytes were capable of complete division, suggesting that these cells still do retain limited proliferative capacity. By quantitatively tracking cardiomyocyte volume dynamics during these proliferative responses, we reveal that both mononucleated and binucleated cells reach a unique size threshold prior to attempted cell division. The absolute threshold is increased by binucleation, which may limit the ability of binucleated cardiomyocytes to divide. By defining the interrelationship between cardiomyocyte size, ploidy, and cell cycle control, we will better understand the cellular mechanisms that drive the loss of mammalian cardiac regenerative capacity after birth.