Surface-initiated polymerization represents a versatile strategy to modify a diverse range of materials with thin, functional polymer coatings. While in many cases the desired functional groups can be directly incorporated via (co)polymerization of the appropriate monomer(s), other functional groups are incompatible with the polymerization strategies that are commonly used to grow polymer brushes and can only be introduced by postpolymerization modification. Determining the local concentration and spatial distribution of these functional groups in postmodified brushes is a challenging task but could help to optimize the design and properties of these polymer coatings. This article reports on the use of X-ray photoelectron spectroscopy (XPS) combined with C-60 cluster ion sputtering to address this challenge. Poly(glycidyl methacrylate) (PGMA) brushes prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) were used as a model platform and were postmodified with propylamine and bovine serum albumin (BSA). The XPS depth-profiling experiments showed that the small propylamine molecules were essentially homogeneously distributed throughout the brush, with the exception of the top few nanometers, which were enriched in propylamine moieties. It was also demonstrated that the amount of propylamine introduced within the polymer brush increased with increasing the postpolymerization reaction time, while no concentration gradients could be observed, indicative of a fast diffusion of the propylamine through the polymer brush layer. On the other hand, XPS depth-profiling experiments performed on polymer brushes that were postmodified with BSA revealed that this protein was only localized in the topmost layers of the polymer coating, which reflects the steric hindrance by the dense polymer brush that prevents efficient diffusion of these large molecules. Together, the results of these experiments demonstrate that XPS depth-profiling combined with C-60 cluster ion sputtering is an efficient and powerful means to study the distribution of functionalities incorporated within a polymer brush layer by postpolymerization modification reactions.