A new in-line method for the monitoring of enzymatic hydrolysis of cellulose is described. Using a new in situ microscope prototype, the noninvasive determination of particle size distributions was possible. For the automated analysis of the acquired images, a new processing algorithm called CelluloseAnalyzer was developed. It enabled tracking of the number of particles and moreover allowed monitoring of the proportions of particle size fractions during the course of enzymatic hydrolysis reactions. Using this technique, significant differences between hydrolysis with endoglucanases and cellulase mixtures were observed. Furthermore, the in situ microscopy results were compared with results from off-line measurements with laser diffraction spectroscopy and gel permeation chromatography.