Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r‐protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r‐proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension‐adapted HEK‐293 and CHO cells: (1) a calcium phosphate based method (Ca‐Pi), (2) a calcium‐mediated method called Calfection, and (3) a polyethylenimine‐based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK‐293 cells in bioreactors. The third method, entirely serum‐free, has been successfully applied to both suspension‐adapted CHO and HEK‐293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK‐293 EBNA cells of some out of more than 20 secreted r‐proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5–10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12‐well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch‐to‐batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.