Adenovirus represents a valuable tool for the treatment of cancer, but tumor targeting remains a pending issue. Most common procedures to modify adenovirus genome are time-consuming due to the requirement of multiple cloning steps, and the low efficacy of the recombination process. Here, we present a new method for homologous recombination in yeast to fast construct recombinant adenoviruses. Also, an alternative procedure to purify viral stocks, based on iodixanol gradient is described. Compared to classical methods, iodixanol is nontoxic to cells, which avoids desalting to use in vitro and in vivo. Moreover, viral stocks are more viable and it can be used for large-scale purifications. Finally, a protocol for analyzing blood persistence of modified vector in in vivo biodistribution is presented.