Polyelectrolyte microparticles synthesized on calcium carbonate cores are considered a promising basis for new drug delivery systems. It is known that microparticles entering a physiological environment absorb proteins on their surface, which can change the properties of the microparticles and alter their functional activity. This study aimed to compare the compositions of the adsorbed protein layer formed on microparticles with the core/shell and shell structures obtained by layer-bylayer deposition. The difference in the microparticle structure was associated with changes in their surface topography and ζ-potential. These microparticles were incubated with human serum or plasma at 37°С for 24 h. The adsorbed proteins were eluted and analyzed by means of SDS-PAGE. The protein composition of the eluates was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results demonstrate that the relative abundance of proteins of different functional groups (immunoglobulins, complement proteins, and apolipoproteins) varied depending on the structure and surface characteristics of the polyelectrolyte microparticles and on the incubation medium. Our findings expand the understanding of the influence of the physicochemical properties of the microparticles on their interaction with proteins, which can help improve the design of microparticles for drug delivery.