Adenoviruses have many attributes, which have made them one of the most widely investigated vectors for gene therapy applications. These include ease of genetic manipulation to produce replication-deficient vectors, ability to readily generate high titer stocks, efficiency of gene delivery into many cell types, and ability to encode large genetic inserts. Recent advances in adenoviral vector engineering have included the ability to genetically manipulate the tropism of the vector by engineering of the major capsid proteins, particularly fiber and hexon. Furthermore, simple replication-deficient adenoviral vectors deleted for expression of a single gene have been complemented by the development of systems in which the majority of adenoviral genes are deleted, generating sophisticated Ad vectors which can mediate sustained transgene expression following a single delivery. This chapter outlines methods for developing simple transgene over expressing Ad vectors and detailed strategies to engineer mutations into the major capsid proteins.