Non-viral vectors based on polyethylenimine (PEI) are usually generated with an excess of PEI. However, the amount of unbound polymer correlates with toxicity limiting the in vivo use of these gene carriers. Purification based on size exclusion chromatography of PEI/DNA polyplexes smaller than 200 nm has been shown to efficiently remove unbound PEI polymer. A novel purification method based on electrophoresis can purify PEI polyplexes independent of their size resulting in polyplexes with final PEI nitrogen/DNA phosphate ratios between 2.6 and 3.1. Also unbound PEI conjugates like PEGylated PEI and transferrin-conjugated PEI can be separated from the polyplexes, providing formulations with clearly defined compositions. Purified polyplexes can mediate in vitro gene transfer with high transfection efficiencies while demonstrating lower cellular toxicity. Purified polyplexes were well-tolerated when systemically delivered into tumor-bearing mice at 100 microg/20 g body weight, with tumor gene expression levels up to 5-fold higher than the non-purified polyplexes. Mice receiving non-purified gene carriers exhibited severe toxicity leading to high mortality and unfavourable gene expression patterns.