Surface modification of drug-loaded particles with polyethylene glycol (PEG) chains is a powerful tool that promotes better transport of therapeutic agents, provides stability, and avoids their detection by the immune system. In this study, we used a new approach to synthesize a biodegradable poly(ester amide) (PEA) and PEGylating surfactant. These were employed to fabricate micro/nanoparticles with a core–shell structure. Nanoparticle (NP)-protein interactions and self-assembling were subsequently studied by synchrotron radiation-based FTIR microspectroscopy (SR-FTIRM) and transmission electron microscopy (TEM) techniques. The core–shell structure was identified using IR absorption bands of characteristic chemical groups. Specifically, the stretching absorption band of the secondary amino group (3300 cm-1) allowed us to identify the poly(ester amide) core, while the band at 1105 cm-1 (C-O-C vibration) was useful to demonstrate the shell structure based on PEG chains. By integration of absorption bands, a 2D intensity map of the particle was built to show a core–shell structure, which was further supported by TEM images.