Recombinant adenoviruses are widely used for delivery and expression of transgenes for therapeutic purposes and in fundamental research. AdEasyTM adenoviral vector system allows for rapid and efficient generation of replication-deficient adenoviruses. One of the crucial steps in producing the recombinant adenoviruses is the generation of a recombinant adenoviral plasmid in E. coli BJ5183 cells via homologous recombination between transgene encoding shuttle vector and pAdEasy-1 plasmid, bearing the greater part of human adenovirus serotype 5 (Ad5) genome. In many published protocols the necessity of electroporation for transformation of the shuttle vector and pAdEasy-1 into E. coli cells is emphasized thus demanding the availability of the appropriate instruments and consumables. Here we proposed the optimized protocol for generation of recombinant adenoviruses with the use of chemically competent E. coli cells only, without the use of electroporation. We showed efficient transformation of linearized non-purified shuttle vector into chemically competent E. coli BJ5183-pAdEasy-1 cells allowing for identification of clones with correct recombinant adenoviral plasmids. Using the optimized protocol we generated four functional replication-deficient adenoviruses, expressing target proteins in HEK293 cells. The proposed protocol allows for significantly simplified and cheaper method for generation of recombinant adenoviruses due to lack of requirement to use instruments and consumables for electroporation.