Standard protein expression systems, such as E. coli, often fail to produce folded, monodisperse, or functional eukaryotic proteins (see Small-scale Expression of Proteins in E. coli). The expression of these proteins is greatly benefited by using a eukaryotic system, such as mammalian cells, that contains the appropriate folding and posttranslational machinery. Here, we describe methods for both small- and large-scale transient expression in mammalian cells using polyethylenimine (PEI). We find this procedure to be more cost-effective and quicker than the more traditional route of generating stable cell lines. First, optimal transfection conditions are determined on a small-scale, using adherent cells. These conditions are then translated for use in large-scale suspension cultures. For further details on generating stable cell lines please (see Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® Recombination Cloning Technology and Flp-In T-REx® lines or Generating mammalian stable cell lines by electroporation).