An objective method to quantify visible cell walls in thin sections of rye dough has been developed using light microscopy. This method is based on the detection of Calcofluor-induced fluorescence of mixed-linked (1,3)(1,4)-β-glucan found in cell walls with a fluorescence microscope, and calculating the area of blue fluorescence in the microscope fields. The method was tested on two rheologically different rye doughs; numerous thin sections were cut from different parts of the dough and from two parallel doughs. A clear difference between the doughs was obtained, supporting results published elsewhere. The method was proven to be reproducible and objective by two test persons. The method is applicable to all biological material containing β-glucan and therefore has potential in, for example, the study of the efficiency of nutrient digestion, in enzyme activity research and in the optimization of processing conditions.