Hypericin is considered to be the most biologically active substance in the crude extract of Hypericum perforatum L. (also known as St. John's wort) and has a wide range of pharmacological effects. In this study, a high resolution high performance liquid chromatography method for determining hypericin was established by comparing different chromatographic conditions. Xylanase-assisted extraction and microwave-assisted extraction can improve the extraction yield of hypericin significantly. And the coupling strategy between two methods resulted in a significant difference on the extraction yield. Microwave-assisted extraction after xylanase-assisted extraction was found to be the most efficient strategy for extracting hypericin. The yield was 0.319 ± 0.006 mg g ⁻¹ , which was a 209.7% increase over unassisted extraction. The combination of enzyme assisted extraction followed by microwave assisted extraction can be more commonly applied to improve extraction efficiency of bioactive compounds from plants.