This article reports the implementation of optical waveguide spectroscopy (OWS) for the quantitative time-resolved observation of changes in the swelling behavior and mass density of protein-functionalized hydrogel films. In the experiment, a thin film of an N-isopropyl-acrylamide (NIPAAm)-based polymer that supported optical waveguide modes is attached to a metallic sensor surface. IgG molecules are in situ immobilized in this gel by using novel coupling chemistry with a charge-attraction scheme based on a tetrafluorophenol sulfonate active ester. The anti-fouling properties of the functionalized hydrogel network and the kinetics of the affinity binding of protein molecules in the gel are investigated.