The application of X-ray photoelectron spectroscopy (XPS) for studies of surface layers of objects with spherical shape was investigated using as examples polystyrene and poly(styrene–acrolein) microspheres with attached human serum albumin (HSA). The amounts of immobilized protein were determined by the standard biochemical Lowry method and by XPS, using the intensity of the N1s signals of HSA as a basis for evaluation. The XPS data were treated by taking into account the spherical shape of the particles analyzed (variable take-off angle of ejected electrons). The best agreement between the results of the biochemical and XPS determinations was found assuming that for the average particle the takeoff angle varies from 0° to 72.7°. This reflects the fact that in the multilayer arrangement of particles, placed onto the support of the XPS apparatus, the particles from the upper layer partially screen the edges of the particles in the layer below.