Natural products, which have been recognized as the most productive source for discovery of new molecular entities, are getting more and more attention for drug discovery. In the research work, high performance liquid chromatography (HPLC) combined with bioassay was used to rapidly discover bioactive compounds from Chinese herbs on the basis of important targets towards to diabetes II (i.e. α-glucosidase) and gout (i.e. xanthine oxidase). Two on-line screening platforms based on post-column bioactivity assay and pre-column affinity assay were developed, respectively. For bioactivity-based screening, some hyphenated techniques coupling HPLC to post-column bioassay were developed to screen and identify the bioactive compounds from complex mixtures without the need of cumbersome purification and subsequent biochemical measurement. For α-glucosidase, three selected beverages were firstly screened by the on-line HPLC-DAD-MS/MS analysis and two potential active compounds with inhibitory activity from Pu-erh tea, EGCG and ECG, were successfully found. Considering the possible false positive inhibition derived from non-specific denatured enzyme, an improved method was subsequently developed with integration of sample preparation and the on-line analysis, and false inhibition caused by tannin greatly decreased. Using this method, two compounds, i.e., corilagin and ellagic acid, with specific α-glucosidase inhibition, were discovered. For xanthine oxidase (XO), two inhibitors including baicalein and wogonin from Scutellaria baicalensis extract were found by on-line fluorescence-based biochemical detection. On account of the XO reaction in vivo, a post-column dual-bioactivity assay was established and applied to measuring the XO inhibitory and free radical scavenging activities of individual compounds in Oroxylum indicum iii extract. For affinity-based screening, an automated on-line restricted-access material solid-phase extraction coupled to column-switching high-performance liquid chromatography (on-line RAM SPE-HPLC) system combined with off-line functional assay was developed for the XO inhibitors screening. The natural product, Oroxylum indicum extract, was analyzed by the developed on-line system, and four compounds with XO inhibition were found and their relative binding capabilities were also ranked. In summary, the two established on-line bioactive screening platforms have been proved to be very useful for rapid profiling and identification of individual active components in complex mixtures, which provided powerful tools for natural product-based drug discovery.