The assessment of cell-surface receptor-ligand interactions is of fundamental importance for understanding plant signal transduction, particularly in immune responses mediated by pattern recognition receptors (PRRs). Iconic biophysical methods such as microscale thermophoresis, isothermal titration calorimetry, and surface plasmon resonance provide precise quantification of binding properties but require specialized equipment, costly reagents, and large quantities of purified proteins or labeled ligands. To offer a more accessible and plant-based approach, this chapter introduces a ligand-depletion binding assay using Nicotiana benthamiana to express PRR extracellular domains (ECDs) and Arabidopsis thaliana seedlings as sensitive biosensors for detecting ligands. Using the ECD of the PRR LORE and its ligand, 3-hydroxydecanoic acid (3-OH-C10:0), as an example, we demonstrate a workflow in which recombinant PRR ECDs are transiently expressed in the apoplast of N. benthamiana via Agrobacterium-mediated transformation. The ligand-binding assay utilizes ultrafiltration by concentrator columns to separate ECD-ligand complexes and unbound ligands by molecular weight. A. thaliana Ca2+ signaling responses serve as a robust readout for ligand detection. This cost-effective and scalable pipeline provides an alternative to conventional methods, enabling medium-throughput screening of receptor variants and ligand candidates. The approach can be broadly adapted to study diverse PRR-ligand interactions, facilitating research in plant immunity.