With the current trend to miniaturize bioactive surfaces to micro-or nanometer scale, the strategy of immobilization becomes more important. Therefore, there is a growing need for protein immobilization techniques that create both stable and homogeneously covered surfaces in a reproducible way. One of the most promising methods to achieve this is the combination of biological receptors with 'click' chemistry, like the Copper catalyzed Alkyne Azide Cycloaddition (CuAAC). This work presents a full optimization of all aspects of the 'click' chemistry reaction between proteins and surfaces in order to create covalently and homogeneously covered biosurfaces. The coupling procedure is monitored by in situ ellipsometry, a unique characterization technique that offers the opportunity to quantify minute amounts of surface coupled protein mass in real-time. The optimization involves the azidification of a solid silicon support, the alkynation of two proteins, Staphylococcus aureus Protein A (SpA) and Maltose Binding Protein (MBP), as well as the development of a highly reproducible CuAAC 'click' coupling protocol. Using the here optimized protocol, active and reproducible biolayers can be created rapidly. The proposed surface biofunctionalization method combined with ellipsometry forms a unique and promising platform towards the development of highly sensitive, accurate biosensors. ; The authors acknowledge financial support from the special research fund (BOF). This study was a part of the Interreg IV-A project "BioMiMedics" (www.nomimedics.org). In the framework of Interreg IV-A, the financial contribution from the EU and the province Limburg (Belgium) is kindly acknowledged. We further acknowledge the financial support from the Interuniversity Attraction Poles Programme (P7/05) initiated by the Belgian Science Policy Office (BELSPO). ; protein surface functionalization; bio-orthogonal chemistry; CuAAC ‘click’ chemistry; ellipsometry; staphylococcus aureus Protein A; Maltose Binding Protein