Plasmodesmata are membrane-lined cytoplasmic passageways that facilitate the movement of nutrients and various types of molecules between cells in the plant. They are highly dynamic channels, opening or closing in response to physiological and developmental stimuli or environmental challenges such as biotic and abiotic stresses. Accumulating evidence supports the idea that such dynamic controls occur through integrative cellular mechanisms. Currently, a few fluorescence-based methods are available that allow monitoring changes in molecular movement through plasmodesmata. In this chapter, following a brief introduction to those methods, we provide a detailed step-by-step protocol for the Drop-ANd-See (DANS) assay, which is advantageous when it is desirable to measure plasmodesmal permeability non-invasively, in situ and in real-time. We discuss the experimental conditions one should consider to produce reliable and reproducible DANS results along with troubleshooting ideas.