Jerica Sabotič and Nika Janež from the Department of Biotechnology of the Jožef Stefan Institute, in collaboration with blue biotechnology experts who are members of the COST Action Network CA18238 Ocean4Biotech, have prepared a comprehensive guide for the use of bioassays for effective discovery of bioactive compounds, which has been published in the prestigious journal Biotechnology Advances. In the search for bioactive compounds from natural resources, bioassays are an indispensable tool that is critical to unlocking optimal bioprospecting potential. This review navigates the complicated landscape of bioassay selection and use and provides valuable insights into the effective exploration of bioactive compounds from natural resources, with a focus on the marine environment. From antimicrobial activity to anti-ageing potential, we explore a spectrum of bioactivities that are critical to the development of pharmaceuticals, food supplements and cosmetics. We emphasize the importance of incorporating various bioassay protocols and performing robust bioassay validation and quality control to ensure the reliability of data generation and safety assessment.