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This paper proposes a new non-invasive, low-cost, and fully automated platform to quantitatively analyze dynamics of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) at the single-cell level by holographic image-based tracking for cardiotoxicity screening. A dense Farneback optical flow method and holographic imaging informatics were combined to characterize the contractile motion of a single CM, which obviates the need for costly equipment to monitor a CM's mechanical beat activity. The reliability of the proposed platform was tested by single-cell motion characterization, synchronization analysis, motion speed measurement of fixed CMs versus live CMs, and noise sensitivity. The applicability of the motion characterization method was tested to determine the pharmacological effects of two cardiovascular drugs, isoprenaline (166 nM) and E−4031 (500 μM). The experiments were done using single CMs and multiple cells, and the results were compared to control conditions. Cardiomyocytes responded to isoprenaline by increasing the action potential (AP) speed and shortening the resting period, thus increasing the beat frequency. In the presence of E−4031, the AP speed was decreased, and the resting period was prolonged, thus decreasing the beat frequency. The findings offer insights into single hiPS-CMs' contractile motion and a deep understanding of their kinetics at the single-cell level for cardiotoxicity screening. • Single cardiomyocytes (CM) motion waveform was generated using holographic image-based tracking method. • Multiple dynamics parameters such as contraction/relaxation speed, contraction/relaxation/resting period were measured. • Verification was carried out with the motion speed measurement of live CMs versus fixed CMs and noise sensitivity analysis. • The beating synchronization between single CMs was tested using the motion waveform generated by the optical flow method. • The CM motion characteristics were compared in control versus drug-treated conditions, other properties were investigated. [ABSTRACT FROM AUTHOR]