Lichens represent a great source of bioactive compounds which are natural in origin. Lichen secondary metabolites are mostly produced by fungal partner, and there exist many reports about their biological and ecological potential. In this chapter, the authors report the whole process for isolation and identification of lichen substances, which is a crucial point to get pure compounds. The content of this chapter includes a short report about lichens used in folk medicine, localization of lichen substances, and history about lichen chemistry. The very valuable part of the chapter is precisely the review about isolation of lichen compounds which were done for the last decades. Description of the whole process which begins from collection of lichen material, thin-layer chromatography (TLC) analysis of present compounds, column chromatography for collecting of fractions, to nuclear magnetic resonance (NMR) spectroscopy as well as high-performance liquid chromatography (HPLC) analysis for identification of compounds is present. In this chapter is also mentioned the importance of physical and chemical properties of lichen compounds.