Agrimony is a traditional Chinese medicine and a herb of Agrimonia pilosa Ledeb belonging to the Rosaceae family. Many pharmacological effects of agrimony have been demonstrated in previous studies. Few studies have screened the active compounds in this complex product for chronic prostatitis. In this study, a two dimensional online method was established. A Sprague-Dawley (SD) rat prostate CMC-online-HPLC/MS method was used to screen, analyze, and identify active compounds acting on α₁ adrenergic receptor from agrimony. Via this online method agrimonolide was screened, analyzed, and identified as the active compound acting on α₁ adrenergic receptor. Thus, agrimonolide is a potential α₁ adrenergic receptor antagonist and requires further study.