Higher plants represent a rich source of new molecules with pharmacological properties, which are lead compounds for the development of new drugs. During the last decades, the renewed interest in investigating natural products has led to the introduction of several important drugs, such as the anticancer drugs vinblastine and taxol, or the antimalarial agent artemisinin. Success in natural products research is conditioned by careful plant selection, based on various criteria such as chemotaxonomic data, information from traditional medicine, field observation, or even random collection. One main strategy in the isolation of new lead compounds consists of so-called bioactivity-guided isolation, in which pharmacological or biological assays are used to target the isolation of bioactive compounds. One major drawback of this strategy is the frequent isolation of known metabolites. Therefore, metabolite profiling using hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR rapidly provides plenty of structural information, leading to a partial or a complete on-line de novo structure determination of the natural products of interest. The combination of metabolite profiling and LC/bioassays gives the possibility to distinguish between already known bioactive compounds and new molecules directly in crude plant extracts (dereplication). Thus, the tedious isolation of compounds of low interest can be avoided and a targeted isolation of new bioactive products. Some examples of rapid localization of bioactive compounds, based on post-chromatographic bioautographic testing of LC/NMR microfractions and subsequent on-line identification will be illustrated in this review. Possibilities and limitations of LC/UV/NMR/MS and LC/bioassay as well as future developments expected in this field will be discussed.