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Summary: Understanding the generation, movement, uptake, and perception of mobile defense signals is key for unraveling the systemic resistance programs in flowering plants against pathogens. Here, we present a protocol for analyzing the movement and uptake of isotopically labeled signaling molecule azelaic acid (AZA) in Arabidopsis thaliana. We describe steps to assess 14C-AZA uptake into leaf discs and its movement from local to systemic tissues. We also detail the assay for uptake and movement of 2H-AZA from roots to the shoot.For complete details on the use and execution of this protocol, please refer to Cecchini et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.