Objective To evaluate the impact of PEG content on poly(lactic‐co‐glycolic acid) (PLGA) NP physicochemical properties, hydrophobic drug release (rifampicin as a model drug) and human serum protein binding. Methods Rifampicin loaded and unloaded nanoparticles with PEG content of 0–17% (w/w) were prepared by an emulsification–evaporation technique. Nanoparticles were characterized for size, zeta potential and morphology. PEGlyation was confirmed using proton nuclear magnetic resonance (1H NMR). Fluorescence spectroscopy and dynamic light scattering were used to determine nanoparticle‐protein binding, binding constants and stability of nanoparticles in human serum, respectively. Drug loading and release were determined by UV‐VIS spectroscopy and drug release data was mathematically modelled. Key findings A NP PEG content of 17% w/w significantly retarded release of rifampicin from PLGA NPs and altered kinetics of drug release. Stern–Volmer (Ksv) protein binding constants decreased upon PEG incorporation. A 2% w/w PEG was sufficient to significantly reduce protein binding extent to PLGA NPs and maintain particle size distributions. Conclusion The ability to fine tune drug release and formation of protein corona around nanoparticles is crucial to formulation scientists. This study suggests that PLGA NPs with low PEG content might be suitable for extended circulation and rapid drug release and that higher PEG content retards hydrophobic drug release.