Cardiovascular disease is a leading cause of morbidity and mortality worldwide. New studies are needed to explore novel therapeutic options for patients that are refractory to existing therapies. Gene transfer using adenoviral vectors has shown promising results in animal studies, and is now being tested in many clinical trials. In this chapter, the advantages of adenoviral vector-mediated gene transfer for cardiovascular disease applications, and the methods on how to construct, propagate, and evaluate adenoviral vectors, are discussed.