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Abstract Plasmodesmata (PD) are highly specialized, nanoscopic pores that traverse the cell wall to connect the cytoplasm of adjacent plant cells, enabling direct cell‐to‐cell communication. PD provides the continuity of three key cellular components: the plasma membrane, the endoplasmic reticulum (ER), and the cytosol. The compressed ER within PD is known as the desmotubule. PD mediates the intercellular trafficking of ions, metabolites, hormones, proteins, and RNA molecules between adjacent cells. Although several methods have been developed to quantify PD‐mediated molecular trafficking, it remains a technical challenge. Among these, PD‐mediated movement of fluorescent proteins is one of the most commonly used approaches. Here we present a microparticle bombardment method using a biolistic particle delivery system to investigate the PD‐mediated movement of fluorescent proteins. We equipped the delivery system with a flow guiding barrel to improve bombardment efficiency and consistency. We demonstrated the effects of gold particle aggregation and plant age on transformation efficiency and protein movement inArabidopsis. We also showed the feasibility of the method in determining PD‐mediated movement in tomato, pepper, and soybean. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Microparticle bombardment assay for measuring plasmodesmata‐mediated trafficking