Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Research investigating the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts as the standard experimental in vitro system. In addition, some studies make use of the H9C2 rat cardiomyoblast cell line, which has the advantage of being an animal-free alternative; however, the extent to which H9C2 cells can accurately mimic the hypertrophic responses of primary cardiac myocytes has not yet been fully established. To address this limitation, we have directly compared the hypertrophic responses of H9C2 cells with those of primary rat neonatal cardiomyocytes following stimulation with hypertrophic factors. Primary rat neonatal cardiomyocytes and H9C2 cells were cultured in vitro and treated with angiotensin II and endothelin-1 to promote hypertrophic responses. An increase in cellular footprint combined with rearrangement of cytoskeleton and induction of foetal heart genes were directly compared in both cell types using microscopy and real-time rtPCR. H9C2 cells showed almost identical hypertrophic responses to those observed in primary cardiomyocytes. This finding validates the importance of H9C2 cells as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell lines for prospective molecular studies in heart development and disease.