Callose (β-1,3-glucan) is both structural and functional component of plasmodesmata (Pd). The turnover of callose at Pd controls the cell-to-cell diffusion rate of molecules through Pd. An accurate assessment of changes in levels of Pd-associated callose has become a first-choice experimental approach in the research of intercellular communication in plants.Here we describe a detailed and easy-to-perform procedure for imaging and quantification of Pd-associated callose using fixed plant tissue stained with aniline blue. We also introduce an automated image analysis protocol for non-biased quantification of callose levels at Pd from fluorescence images using ImageJ. Two experimental examples of Pd-callose quantification using the automated method are provided as well.