Objective: The purpose of this study was to develop and validate a specific and sensitive liquid chromatography tandem mass-spectrometry method for quantification of usnic acid concentration in the lichen, Cladonia uncialis, suitable for detection of relatively small fluctuations of usnic acid concentration in response to environmental changes. Results: The resulting method was fully validated according to international guidelines and demonstrated good selectivity and sensitivity with minor levels of a matrix effect and high accuracy. Cladonia uncialis, LC-MS, MS, Lichen, Liquid chromatography tandem mass-spectrometry, Secondary metabolites, Usnic acid