Heartbeat measurement is important in assesssing cardiac function because variations in heart rhythm can be the cause as well as an effect of hidden pathological heart conditions. Zebrafish (Danio rerio) has emerged as one of the most useful model organisms for cardiac research. Indeed, the zebrafish heart is easily accessible for optical analyses without conducting invasive procedures and shows anatomical similarity to the human heart. In this study, we present a non-invasive, simple, cost-effective process to quantify the heartbeat in embryonic zebrafish. To achieve reproducibility, high throughput and flexibility (i.e., adaptability to any existing confocal microscope system and with a user-friendly interface that can be easily used by researchers), we implemented this method within a software program. We show here that this platform, called ZebraBeat, can successfully detect heart rate variations in embryonic zebrafish at various developmental stages, and it can record cardiac rate fluctuations induced by factors such as temperature and genetic-and chemical-induced alterations. Applications of this methodology may include the screening of chemical libraries affecting heart rhythm and the identification of heart rhythm variations in mutants from large-scale forward genetic screens. T he zebrafish (D. rerio) is a model organism that has rapidly reached a respected position in biomedical research 1,2 . The zebrafish has traditionally been used as a model for developmental biology because of its many advantages, such as its rapid external development, the large number of eggs produced by a single cross, and the ease of genetic manipulation 3,4