This thesis explores the use of high-content image analysis to assess maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Cardiovascular diseases, such as myocardial infarction and heart failure, demand reliable in-vitro models, yet current hPSC-CMs remain functionally immature, limiting their application in research and therapy. Maturation strategies, including biochemical and biophysical cues, long-term culture, and optimized media, have shown potential but require standardized, high-throughput methods to assess effectiveness. Here, a novel morphological profiling approach incorporating classifier models quantified maturation-related features in hPSC-CMs cultured under varying conditions. The distinct maturation phenotypes of these hPSC-CMs were demonstrated with global proteomic cluster analysis. The developed morphological assay effectively differentiated maturation states across different culture conditions, offering a potential tool for screening in vitro systems that facilitate hPSC-CM maturation. This advancement enhances the translational utility of these cells in cardiovascular research.