Key message Liquid chromatography is the main method for assaying bioactive compounds in biomass. Total phenolic content and antioxidant capacity are measured with spectroscopy. Phenolics are the most important bioactive substances in short rotation coppice. Secondary metabolites of black locust, willow and poplar have many different activities. The richest source of bioactive compounds are flowers, bark and buds. Abstract The black locust (Robinia pseudoacacia), willow (Salix spp.) and poplar (Populus spp.) may be the source of the biomass obtained from natural habitats as well as intentionally established plantations in the system of short rotation coppice. These species are promising candidates as an alternative source of bioactive compounds in researches for developing new bioproducts for multidirectional uses, including polymers, lubricants, construction materials, pharmaceuticals, as well as bioenergy and fuels. Various parts of plants, such as leaves, flowers, seeds, bark, woods, buds, catkins, whole tree material and bee products (honey, propolis) are a source of bioactive compounds. The characterization of these compounds, especially phenolic compounds (flavonoids, stilbenes, lignans, phenolic acids, tannins and phenolic glucosides), fatty acids, sterols, etc. may be determined by a variety of analytical (spectroscopy, chromatography) and extraction methods. This review contains 131 references and systematize all available data on the characterization of the bioactive compounds in the biomass of black locust, poplar and willow by analytical and extraction methods.