In the model plant Arabidopsis, the best studied Pattern-triggered immunity (PTI) system is perception of the bacterial pathogen-associated molecular pattern (PAMP) flagellin, or its active peptide-derivative flg22, by the plasma membrane-localized receptor FLAGELLIN SENSING 2 (FLS2). Flg22 perception initiates an array of immune responses including the fast and transient production of reactive oxygen species (ROS). In addition, FLS2 undergoes ligand-induced endocytosis and subsequent degradation within 60 min of flg22-treatment. Luminol-based assays are routinely used to measure extracellular ROS production within minutes after flg22 treatment. Many mutants in flg22-response pathways display defects in flg22-induced ROS production. Here, we describe a luminol-based ROS Re-elicitation Assay that can be utilized to quantitatively assess flg22-signaling competency of FLS2 at times during which FLS2 is internalized, trafficked through endosomal compartments, and degraded in response to flg22. This assay may also be employed to correlate FLS2 signaling competency with receptor accumulation in vesicular trafficking mutants that either affect FLS2 endocytosis or replenishment of FLS2 through the secretory pathway. In addition, this assay can be extended to studies of other PAMP (ligand)-receptor pairs.