Contraction of muscle reflects its physiological state. Methods to quantify contraction are often complex, expensive and tailored to specific models or recording conditions, or require specialist knowledge for data extraction. Here we describe an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. MUSCLEMOTION allowed rapid and easy measurement of contractility in (i) single cardiomyocytes from primary adult heart and human pluripotent stem cells, (ii) multicellular 2D-cardiomyocyte cultures, 3D engineered heart tissues and cardiac organoids/microtissues in vitro and (iii) intact hearts of zebrafish and humans in vivo. Good correlation was found with conventional measures of contraction in each system. Thus, using a single method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell- and animal models and human echocardiograms.