Abstract: This work describes the development of a characterization method for biofunctionalized surfaces and its use for biosensor applications. The method is based on the processing of fluorescence images obtained by confocal microscopy. It retrieves quantitative data about the quantity and distribution of surface bound biomolecules, used to improve a biosensor performances in terms of specificity and sensitivity. Two biofunctionalization protocols were compared using the developed tools based on the fluorescence intensity, the surface coverage and the homogeneity of repartition of the fluorescence. In addition, the stability of the layer under flow, as well as the reproducibility and robustness of the protocols were evaluated.