Optical waveguide lightmode spectroscopy (OWLS), based on the incoupling of laser light into a waveguide sensor by an optical grating, allows for the in situ measurement of protein adsorption. Few reports have described cleaning methods for the surfaces of such sensors, and in this investigation, we compare common methods for cleaning of silica surfaces in relation to their effectiveness for cleaning silica‐coated waveguide sensors used in OWLS. For this purpose, atomic force microscopy (AFM) analysis of surface morphology and OWLS detection of protein adsorption kinetics were used to evaluate waveguide sensors before and after cleaning. While AFM line scans showed a substantial increase in average waveguide peak‐to‐valley height after RCA cleaning relative to all other methods tested, chemical etching owing to the alkaline component of the rolling circle amplification method rendered the waveguide unusable for detection of protein adsorption with OWLS. A revised method, based on replacement of the alkaline step with immersion in sodium dodecyl sulfate, was not only effective at cleaning OWLS waveguides off‐the‐shelf but also showed excellent protein adsorption reproducibility after ex situ cleaning. Moreover, the revised method showed excellent reproducibility when applied in situ, between repeated adsorption‐elution cycles. Copyright © 2013 John Wiley & Sons, Ltd.