Transduction of therapeutic transgenes using multiply attenuated viral vectors is considered an essential technology for gene therapy scenarios. While first-generation viral transduction systems were engineered for constitutive expression of a single therapeutic transgene, most advanced viral gene-transfer technologies enable regulated expression of several transgenes. Efficient transfer of numerous transgenes enables co-expression of therapeutic transgenes along with marker or selection determinants, production of multi-subunit protein complexes, or combinatorial expression of a particular set of genes to treat multigenic disorders. Likewise, adjustable transcription control is fundamental to adapt therapeutic protein production to the changing daily dosing regimes of a patient, to titrate expression of protein pharmaceuticals into the therapeutic window, and to reverse dosing upon completion of the therapy. Also, conditional transcription dosing has been successfully used for production of difficult-to-express protein therapeutics in biopharmaceutical manufacturing and for sophisticated gene-function analysis in basic research programs. By way of example, we provide detailed design (auto-regulated and binary dual-regulated expression configurations), production (generation, purification, and quality control of transgenic adenovirus particles), and handling (transduction) protocols for adenovirus vectors that enable transduction of mammalian cells for regulated expression of several transgenes.