Over the past two decades, bioactivity-guided drug screening has become a pivotal approach in drug discovery, which could identify potential drug candidates with desirable therapeutic effects against specific disease phenotypes or targets. This strategy has led to the discovery of many clinical medications such as natural products, peptides, and antibody drugs, and become a mainstream method. Herein, we first systematically summarize the principles and applications of recent bioactivity-guided drug screening strategies through technological advances in in silico screening, cell model systems, organoid model systems, and animal model systems. We then provide an overview on the representative active compounds, pre-clinical drugs, and marketed drugs, and discuss the present challenges and future directions of bioactivity-guided drug screening methods for clinical drug development. Based on the research advancements, it is anticipated that more disease-relevant screening models and efficient methods can be developed to identify drugs with ideal therapeutic effects and favorable pharmacological properties.