Insulin (INS) was encapsulated into complexes with poly(ethylene glycol)-block poly(L-lysine) (PEG-b-PLys), which is a polypeptide-based block copolymer (a neutral-cationic block polyelectrolyte). These macromolecules can encapsulate INS molecules in aqueous conditions via electrostatic interactions. Light scattering techniques are used in order to examine the complexation process of the hybrid nanoparticles in a gamut of buffers, as a function of protein concnetration. The physicochemical and structural characteristics of the complexes depend on the ionic strength of the aqueous medium, while the concentration of PEG-b-PLys was constant through the series of solutions. As INS concentration increased each polyelectrolyte chain interacts with an increasing number of INS molecules, the degree of charge neutralization becomes higher and the size distribution of the complexes decreased also, especially at the highest ionic strength. The size/structure of complexes diluted in biological medium indicated that the copolymer imparts stealth properties and colloidal and biological stability to the complexes, which could in turn affect the clearance properties in vivo. Therefore, these studies could be a rational roadmap for designing the optimum complexes/effective nanocarriers for proteins and peptides.