The mechanical performance of cardiomyocytes is an important indicator of their maturation state and of primary importance for the development of therapies based on cardiac stem-cells. As the mechanical analysis of adherent cells at high-throughput remains challenging, we explore the applicability of real-time deformability cytometry (RT-DC) to probe cardiomyocytes in suspension. RT-DC is a microfluidic technology allowing for real-time mechanical analysis of thousands of cells with a throughput exceeding 1,000 cells per second. For cardiomyocytes derived from human induced pluripotent stem cells, we determined a Young’s modulus of 1.250.08 kPa which is in close range to previous reports. Upon challenging the cytoskeleton with cytochalasin D (CytoD) to induce filamentous actin depolymerization, we distinguish three different regimes in cellular elasticity. Transitions are observed below 10 nM and above 103 nM and are characterized by a decrease in the Young’s modulus. These regimes can be linked to cytoskeletal and sarcomeric actin contributions by cardiomyocyte contractility measurements at varying CytoD concentrations, where we observe a significant reduction in pulse duration only above 103 nM while no change is found for compound exposure at lower concentrations. Comparing our results to mechanical cell measurements using atomic force microscopy we demonstrate for the first time the feasibility of using a microfluidic technique to measure mechanical properties of large samples of adherent cells while linking our results to the composition of the cytoskeletal network.