This work presents a method to quantify the lignification of maize tissues by automated color image analysis of stained maize stem cross sections. Safranin and Alcian blue staining makes lignified tissues appear red, and nonlignified tissues appear blue. Lignification is assessed by the ratio of red intensity over blue intensity. A rough quantification of global lignification is computed as the surface ratio of lignified tissues. A more precise quantification is obtained by computing profiles of red/blue intensity ratio in relation to the distance to the epidermis, depicting the spatial distribution of lignified walls within the stem. Lignification profiles are analyzed through summary parameters describing the evolution of lignification in three specific regions. The distribution of lignification can be quickly assessed depending on the position and the development stage, allowing the screening of genetic variations to be envisioned.