AbstractPoly(ethylene imine) can be considered as the gold standard for DNA delivery into cells in vitro, but severe cytotoxic side‐effects and inapplicability for targeted approaches in vivo urgently call for the design of new gene carriers. Since poly(2‐oxazoline)s (P(Ox)s) can be easily synthesized and modified, this polymer class might be ideal for the optimization of polymeric transfection processes. The utilization of 2‐methyl‐2‐oxazoline (MeOx) and 2‐ethyl‐2‐oxazoline (EtOx) is also known to be beneficial because these monomers were suggested to overcome solubility issues, mediate stealth behavior and, consequently, facilitate a reduction of cytotoxicity. A series of amino (AmOx) functionalized P(Ox) copolymers with either MeOx (gradient copolymers) or EtOx (random copolymers) was synthesized, deprotected and biochemically characterized regarding cytotoxicity, polyplex formation ability, cellular uptake, and transfection efficiency. Polymers with percentages of AmOx higher than 35 mol % showed stable polyplex formation and also an increase in cytotoxicity. All elucidated P(Ox)s revealed a poor transfection efficiency in both L929 and Hepa1‐6 cell lines. However, the investigations contribute to the understanding of the influence of stealth units (MeOx and EtOx) and their distribution within the polymer chain on selected properties of polyplexes and describe characteristics of amino functionalized P(Ox)s in different cell lines.