Isolated, perfused hearts are viable for hours outside the body, and important research findings have been made using mouse hearts ex vivo. In the Langendorff perfusion mode, the coronary tree is perfused via retrograde flow of a perfusate down the ascending aorta. Although the Langendorff setup is generally simpler and quicker to establish, the working heart mode allows the heart to function in a more physiologically relevant manner, where the perfusate is directed into the left ventricle via the left atrium. The contracting, fluid-filled ventricle will eject the perfusate into the aorta in a more physiologically relevant manner, lifting the physiological relevance of the contractile and energetic data. The workload on the heart (preload, afterload and heart rate) can be precisely adjusted in the working, isolated heart, and the ventricular performance, for example, end-diastolic and end-systolic pressures, stroke volume, cardiac output, and oxygen consumption can be determined. Moreover, using pressure-volume catheters, ventricular performance can be assessed in great detail. With the present review, we highlight the benefits and drawbacks of the technique and indicate where particular attention must be put when building the working heart setup, designing experiments, executing the studies, and analyzing the obtained data.