In this work we report a one-step method for the fabrication of poly(ethylene glycol) PEG-like chemical gradients, which were deposited via continuous wave radio frequency glow discharge plasma polymerization of diethylene glycol dimethyl ether (DG). A knife edge top electrode was used to produce the gradient coatings at plasma load powers of 5 and 30 W. The chemistry across the gradients was analyzed using a number of complementary techniques including spatially resolved synchrotron source grazing incidence FTIR microspectroscopy, X-ray photoelectron spectroscopy (XPS) and synchrotron source near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Gradients deposited at lower load power retained a higher degree of monomer like functionality as did the central region directly underneath the knife edge electrode of each gradient film. Surface derivatization experiments were employed to investigate the concentration of residual ether units in the films. In addition, surface derivatization was used to investigate the reactivity of the gradient films toward primary amine groups in a graft copolymer of poly (L-lysine) and poly(ethylene glycol) (PLL-g-PEG copolymer) which was correlated to residual aldehyde, ketone and carboxylic acid functionalities within the films. The protein adsorption characteristics of the gradients were analyzed using three proteins of varying size and charge. Protein adsorption varied and was dependent on the chemistry and the physical properties (such as size and charge) of the proteins. A correlation between the concentration of ether functionality and the protein fouling characteristics along the gradient films was observed. The gradient coating technique developed in this work allows for the efficient and high-throughput study of biomaterial gradient coating interactions.