Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractive to analysis due to the prevalent high degree of non-specific binding. In this manuscript we explore the dynamic process of the formation of self-assembled monolayers and optimize physical and chemical properties thus reducing considerably non-specific binding while maintaining the integrity of the immobilized biomolecules. As a result, analysis of specific binding of analytes to immobilized target molecules is significantly facilitated.