With chemical analysis and bioassay testing, information can be obtained about the composition and biological activity of a sample. Because chemical analysis on its own does not return information about bioactivity and bioassay testing of a sample, the extract does not show which compound is responsible for the measured activity. A combination of chemical analysis and biotesting can be a powerful approach for the identification of unknown and emerging bioactives. This is done via so‐called effect‐directed analysis (EDA) or bioassay‐guided fractionation (BGF). In these approaches fractionation is used to reduce the sample complexity down to a few compounds or a single compound per fraction, which allows the pinpointing of bioactives. This chapter discusses sample preparation, fractionation techniques, bioassays, and chemical analysis as well as identification strategies for the research fields of environmental analysis, food analysis, and drug discovery.