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Recombinant protein-based biologics are increasingly relevant in the pharmaceutical industry. Currently, mammalian cell-based bioprocesses are a routine manufacturing procedure for complex proteins such as recombinant antibodies, and the number of new biologics produced by mammalian cells is progressively increasing. In addition, a growing number of proteins need to be rapidly screened to identify new candidates for clinical trials. To help fill this need, rapid mammalian-based bioprocesses have been established with transient gene expression, the production of recombinant protein following gene delivery into cells without the establishment of a stable cell line. With current transient gene expression methods, the recombinant protein can be made available in milligram amounts within 1–2 weeks from the time the appropriate expression vector has been generated. Thus, pre-clinical material can quickly enter the drug screening process (in vitro activity tests and animal studies), and successful candidates can be identified within a short time-frame. But the potential of this innovative technology is still far from being fully exploited and appreciated. The aim of this article is to provide an overview of the past achievements and current status of the field of transient gene expression in mammalian cells as well as to discuss the future perspectives and challenges of this promising technology