Lichens are a large group of valuable lower plants with unique features and diverse applications worldwide such as in medicine, cosmetics, food, and textile industries. They are also well known for their potential in observing climate and environmental monitoring. Their successful exploitations require reliable analytical methods to check and maintain the quality and efficacy of the products based on them. Several mass spectrometry-based methods utilizing GC–MS, LC–MS, MALDI–MS, etc., were reported for the efficient analysis of lichen metabolites. Among them, LC–MS analytical platforms by combining the strengths of liquid chromatography with the capabilities of mass spectrometry provide better sensitivity, selectivity, high-throughput, and universal detection to the wide-ranging molecules of lichen compounds. The applications of LC–MS in lichen research including screening for the dereplication, lichen metabolomics, metabolite profiling, and absolute quantifications, are summarized in this chapter with examples.