Cardiovascular diseases, including heart failureHeart failure and myocardial infarction, are leading causes of morbidity and mortality worldwide. Cardiotoxicity, a major concern in drug development, further emphasizes the need for accurate in vitro models for testing the potential drug candidates. The cardiac-specific cells are integral to the successful design of in vitro cardiotoxicity assessment models. Traditional two-dimensional (2D) systems and animal models fail to replicate the complex human cardiac environment, driving the development of three-dimensional (3D) models. These models, incorporating cardiomyocytes, fibroblasts, endothelial cells, and biomaterials including hydrogels, more accurately mimic human heart tissue. In this chapter, we explore the role of heart-derived stromal cells (HSCs) in cardiotoxicity assessment using in vitro 3D models, highlighting their advantages over traditional methods. The integration of in vitro 3D models into drug discovery/testing using HSCs promises the predictive accuracy of cardiotoxicity testing and improve therapeutic outcomes.