Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSC-CM immaturity by introducing a physiologically relevant micropatterned substrate for long-term culture and maturation. An innovative microfabrication methodology combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue. Platinum electrodes were integrated into the cell culture chamber enable pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca2+ transient, sarcomere orientation, and nucleus aspect ratio were analyzed in a 2D hiPSC-CM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSC-CMs for three weeks on a micropatterned PDMS substrate (2.5-5 µm deep, 20 µm center-to-center spacing of grooves, 2-5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, contractility, and cytosolic Ca2+ transient. The study provides insights into substrate stiffness effects on hiPSC-CM contractility and Ca2+ transient at immature and mature states. Maximum contractility and fastest Ca2+ transient kinetics occur in mature hiPSC-CMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. MS proteomic analysis further revealed that hiPSC-CMs cultured on soft micropatterned substrates exhibit advanced maturation, marked by significant upregulation of key structural, electrophysiological, and metabolic proteins. This new substrate offers a promising platform for disease modeling and therapeutic interventions. STATEMENT OF SIGNIFICANCE: Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have been transformative to disease-in-a-dish modeling, drug discovery and testing, and autologous regeneration for human hearts and their role will continue to expand dramatically. However, one of the major limitations of hiPSC-CMs is that without intervention, the cells are immature and represent those in the fetal heart. We developed protocols for the fabrication of the PDMS matrices that includes variations in its stiffness and micropatterning. Growing our hiPSC-CMs on matrices of comparable stiffness to a healthy heart (5 kPa) and grooves of 20 μm, generate heart cells typical of the healthy adult human heart.