The most efficient system to introduce genes of interest within the adenovirus genome is by homologous recombination in microorganisms. In this chapter, the most popular procedures are described: two for homologous recombination in Escherichia coli, and one in yeast. Main differences between procedures are found in the plasmids needed as well as in the selection system used to rapidly identify newly generated recombinant adenovirus. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized to generate a viral pre-stock in permissive human cells. Finally, as a previous step before its amplification at medium or large scale, the viral pre-stock must be analyzed to quantify its potency and infectivity as well as to exclude the presence of unwanted replication competent particles.