Understanding of the interfacial chemistry of ultrathin polymeric adlayers is fundamentally important in the context of establishing quantitative design rules for the fabrication of nonfouling surfaces in various applications such as biomaterials and medical devices. In this study, seven poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-PMOXA) copolymers with grafting density (number of PMOXA chains per lysine residue) 0.09, 0.14, 0.19, 0.33, 0.43, 0.56, and 0.77, respectively, were synthesized and characterized by means of nuclear magnetic resonance spectroscopy (NMR). The copolymers were then adsorbed on Nb2O5 surfaces. Optical waveguide lightmode spectroscopy method was used to monitor the surface adsorption in situ of these copolymers and provide information on adlayer masses that were then converted into PLL and PMOXA surface densities. To investigate the relationship between copolymer bulk architecture (as shown by NMR data) and surface coverage as well as surface architecture, time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was performed. Furthermore, ToF-SIMS method combined with principal component analysis (PCA) was used to verify the protein resistant properties of PLL-PMOXA adlayers, by thorough characterization before and after adlayer exposure to human serum. ToF-SIMS analysis revealed that the chemical composition as well as the architecture of the different PLL-PMOXA adlayers indeed reflects the copolymer bulk composition. ToF-SIMS results also indicated a heterogeneous surface coverage of PLL-PMOXA adlayers with high grafting densities higher than 0.33. In the case of protein resistant surface, PCA results showed clear differences between protein resistant and nonprotein-resistant surfaces. Therefore, ToF-SIMS results combined with PCA confirmed that the PLL-PMOXA adlayer with brush architecture resists protein adsorption. However, low increases of some amino acid signals in ToF-SIMS spectra were detected after the adlayer has been exposed to human serum.