The surface concentration of CBD-FITC conjugates, adsorbed on cellulose fibers, was determined by image analysis. The program consists of two scripts, the first dedicated to the elaboration of the calibration curve. The emission of fluorescent light, detected by image analysis, is correlated with the concentration of CBD solutions. This calibration is then used (second script) to determine the concentration of CBDs adsorbed on cellulosic fibers. This method allows the direct estimation of the surface concentration of adsorbed CBDs, which usually is not accurately calculated from depletion studies, since the surface area is hardly known. By observing different spots in the surface of the fibers, site-specific information is obtained. It was verified that the physically heterogeneous fibers exhibit different amounts of adsorbed CBDs.