The mammalian genome contains thousands of regulatory elements whose activities are highly sensitive to external and internal stimulation. Here, we present a protocol to assess the murine cardiac function of regulatory elements using an adeno-associated virus serotype 9 (AAV9)-based massively parallel reporter assay (MPRA). We describe steps for the selection principle of enhancer candidates, MPRA library construction, animal delivery, sequencing library generations, and data analysis pipeline. Our MPRA protocol provides a comprehensive workflow for enhancer activity quantification in vivo. For complete details on the use and execution of this protocol, please refer to Yangpo Cao et al.(1).