Coordinated and synchronous contraction of cardiomyocytes ensures a normal cardiac function while deranged contraction of cardiomyocytes can lead to heart failure and circulatory dysfunction. Detailed assessment of the contractile property of cardiomyocytes not only helps elucidate the pathophysiology of heart failure but also facilitates development of novel therapies. Herein, we report application of atomic force microscopy to determine essential mechanodynamic characteristics of self-beating cardiomyocytes including the contractile amplitude, force, and frequency. The contraction was continuously measured on the same point of the cell surface; the result assessed postintervention was then compared with the baseline, and the fractional change was obtained. We employed short-time Fourier transform to analyze the time-varying contractile properties and calculate the spectrogram, based on which subtle dynamic changes in the contractile rhythmicity were delicately illustrated. To demonstrate potential applications of this approach, we examined the inotropic and chronotropic responses of cardiomyocyte contraction induced by various pharmacological interventions. The administration of epinephrine significantly increased the contractile amplitude, force, and frequency whereas esmolol markedly decreased these contractile properties. As uniquely illustrated in the spectrogram, doxorubicin not only impaired the contractility of cardiomyocytes but also drastically compromised the rhythmicity. We envision that our approach should be useful in research fields that require detailed evaluation of the mechanodynamic response of cardiomyocytes, for example, to screen drugs that possess cardiac activity or cardiotoxicity, or to assess chemicals that could direct differentiation of stem cells into functioning cardiomyocytes.