This study describes the first example for shielding of a high performing terpolymer that consists of N‐(2‐hydroxypropyl)methacrylamide (HPMA), N‐(3‐guanidinopropyl)methacrylamide (GPMA), and N‐(2‐indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative – poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO₉MA)) via reversible addition–fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO₉MA) is varied from 3 to 40 kg mol^–1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO₉MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO₉MA) block demonstrates excellent biocompatibility with higher cell viability in L‐929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO‐K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO₉MA) up to 10 kg mol^–1. Moreover, a fluorescently labeled analogue of P(MEO₉MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol^–1 of P(MEO₉MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).