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We present concise results of method validation for trehalose quantitation by LC-MS/MS in spruce ectomycorrhizal roots in order to describe spruce health status, mainly in connection to contamination by a pathogenic fungus, Gemmamyces piceae. The procedure is based on Rogatsky et al. (2005) developed for human plasma. We found out that the best extraction yield was achieved with 80% methanol/water (v/v) solution and optimal extraction temperature was set between 50-60°C. In contrast to previous papers, we minimized the activity of trehalase enzyme by putting root samples into liquid N2 immediately after root excavation, followed by freeze-drying in order to stop trehalase activity. Higher content of trehalose was recorded in healthy trees, confirming the idea that ectomycorrhiza plays a significant role in plant-pathogen interactions.