RATIONALE: Understanding plant defense mechanisms against pathogens is critical for agricultural productivity and crop protection. This study focuses on the quantification of camalexin and scopoletin, essential phytoalexins in Arabidopsis thaliana, using advanced mass spectrometry techniques. Accurate measurement of these compounds can provide insights into plant resistance and support agricultural research. METHODS: Camalexin and scopoletin were quantified using matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS). The matrix and solvent conditions were optimized to enhance sensitivity and accuracy. MS/MS experiments confirmed the identification with high mass accuracy (mass error < 5 ppm). RESULTS: The method demonstrated high linearity for scopoletin (R ² = 0.9992) and camalexin (R ² = 0.9987) over concentration ranges of 0.16-5 µM and 0.31-5 µM, respectively. The limits of detection (LOD) were 0.16 µM for camalexin and 0.04 µM for scopoletin, while the limits of quantification (LOQ) were 0.31 µM for camalexin and 0.16 µM for scopoletin. The average relative standard deviation was 1.43% for scopoletin and 2.46% for camalexin, with average relative errors of 3.91% and 4.11%, respectively. CONCLUSIONS: This study presents a precise, and accurate method for the quantification of key phytoalexins in Arabidopsis thaliana. The developed MALDI-HRMS approach significantly contributes to the understanding of plant defense mechanisms and offers potential applications in agricultural and biotechnological research.