Ethylene is a gaseous phytohormone that plays an important role as a signaling molecule during pathogen attack, influencing disease resistance and defense responses in plants. A precise measurement of ethylene production upon pathogen challenge is essential to elucidate its role in plant–pathogen interactions. Gas chromatography (GC) is among the most accurate and sensitive techniques for detecting and quantifying ethylene emissions due to its selectivity and effectiveness with gaseous molecules. In this chapter, we provide a detailed procedure employing GC specifically adapted for measuring ethylene levels during pathogen infection (Botrytis cinerea) in Arabidopsis leaflets. Arabidopsis leaflets infected by the necrotrophic pathogen Botrytis cinerea exhibit increased ethylene emission, facilitating the activation of defense pathways and secondary metabolites such as camalexin. The present GC method captures ethylene dynamics at early infection stages, ensuring precise quantification critical for dissecting the molecular mechanisms of plant immunity.