In cardiovascular research, diverse ex vivo models are used to investigate cardiac function. These models can be categorized according to their complexity, ranging from isolated cardiomyocytes to multicellular 3-dimensional tissue preparations, such as the Langendorff-perfused heart or coronary-perfused wedges. Cardiac tissue slices bridge the gap between these models, as their relatively low thickness overcomes the need for arterial perfusion, while the native cellular alignment and extracellular matrix structure are preserved. This enables the use of tissue when coronary perfusion is not available (e.g., tissue excised during surgery for congenital heart disease). The present protocol describes the preparation of viable cardiac slices from myocardial explants from neonate and infant patients undergoing surgery for congenital heart disease. Upon extraction, the myocardial tissue is transferred to oxygenated, ice-cold, low-calcium solution and transported to the laboratory. Thereafter, the tissue is pre-cut, embedded into low-melting agarose, and sectioned with a vibratome. Tissue recovery is promoted by the stepwise increase of calcium concentration, followed by gradual rewarming to 37 °C for 1 h in the measurement solution. Afterwards, the obtained acute myocardial slices can be used for physiological experiments. Representative results for isometric force measurements and action potential recordings are provided. The importance of the solution and the vibratome parameters to the preparation of viable cardiac slices, as well as limitations regarding the control of the fiber alignment and long-term culture, are discussed.