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Medicinal plants are of great interest as starting material for identification of new biologically active compounds. A large number of low-molecular-weight compounds isolated from plants or microorganisms have already been identified as effective in diseases as diverse as HIV infection, herpes simplex, neuroblastoma, and breast cancer. Some drugs that emerged from this process are already in the late-phase clinical trials. The first step for the identification of bioactive compounds in the biomedical field is the screening of extracts from different tissues of several medicinal plants for a given activity (for instance, in vitro antiproliferative activity). To this aim, the method of extraction of bioactive compounds is crucial. The second step is the fractionation and the characterization of the plant extracts exhibiting the desired biological activity. This step takes advantage from several analytical and preparative procedures, among which preparative and analytic high-performance liquid chromatography (HPLC), several HPLC-based methods, such as HPLC/MS, gas chromatography/mass spectrometry (GC/MS), surface plasmon resonance (SPR)-based biospecific interaction analysis (BIA) employing biosensors. Applications of these methodologies to the screening, identification, purification, and characterization of bioactive compounds from medicinal plants are described in this review. [ABSTRACT FROM AUTHOR]