Abstract: Affinity ultrafiltration mass spectrometry (AUF-MS) technology is a rapid, simple and effective method for the discovery and development of small active molecule in traditional Chinese medicine (TCM). This technology was initially developed in the middle of 1990s, which was introduced to target-oriented drug discovery. Through the ligand-receptor specific binding characteristics, the affinity ultrafiltration device can facilitate the rapid screening of small-molecule ligands from the complex extracts, and high performance liquid chromatography-mass spectrometry (HPLC-MS) assists in the structural identification of potentially active small drug molecules. Thus, AUF-MS has become a powerful tool for identifying bioactive molecules from complex chemical matrixes. The general workflow of AUF-MS is not complicated: depending on the molecular weight cut-off of the semi-permeable membrane, the ligand-bound protein complexes are retained by the membrane under centrifugal force to separate the bound and unbound components of the analyte. Bound ligands are eluted from the ultrafiltration membrane by destabilizing the target-ligand complex with an organic solvent or pH change, and then the ligands are consequently subjected to analysis by LC-MS to identify and characterize the low molecular weight compounds that interact with target molecules. The classic strategies of screening active ingredients from natural product, such as the high-throughput screening methods based on ultraviolet fluorimetric or radioactive detection, the traditional phytochemical screening procedure (i.e. isolation, structure elucidation and bioactivity test), are not able to meet the urgent need of contemporary pharmacology research due to its time consumption and a high rate of false positive or negative results. In comparison with those methods, AUF-MS is easier to be operated without unnecessary isolation or purification of inactive compounds and directly screen bioactive ingredients from the Chinese herb. Also, only modest amounts of target proteins are required, which are no labeling or immobilization. AUF-LC-MS was employed to identify inhibitors of enzymes such as α-glucosidase, xanthine oxidase, neuraminidase from Chinese herb medicine. The results showed that AUF-MS performed better reproducibility and more tolerance of interferences than classic strategies for certain targets. In this review, Firstly, a brief introduction was exhibited about the general procedures of AUF-MS, and the key factors affecting the result of AUF-MS, including the selection of ultrafiltration membrane and dissociation, the dose of receptors. Secondly, the recent applications of AUF-MS to screen potential bioactivity small molecules from the natural product in the past ten years were summarized. These results based on AUF-MS to screen active compounds were expected to be valuable for discovering drug molecules candidates from TCM and efficiently designing drugs for prevention and the treatment of diseases. Finally, the future prospects of AUF-MS were also presented. AUFMS technology is expected to provide new research ideas for the screening of active components from TCM.