Benign prostatic hyperplasia (BPH) is a common disease in elderly men. The main treatment for BPH is α-adrenergic antagonists and 5α-reductase inhibitors. In this study, a two-dimensional (2D) α1A cell membrane chromatography (CMC) online liquid chromatography/mass spectrometry system was built. Fructus Piperis, a traditional Chinese medicine and food homolog, was assayed with this 2D system. Piperine was identified as the active compound acting on α1A receptors. A competitive binding assay and molecular docking assay were performed to investigate the binding sites and the affinity of piperine for the α1A receptor. The results of the competitive binding assay (dissociation equilibrium constant of tamsulosin was 1.43 × 10−6 M and piperine was 2.13 × 10−6 M) and molecular docking assay (total score for tamsulosin binding with the α1A receptor was 6.9719, and for piperine it was 4.4891) corresponded with the retention time of tamsulosin and piperine on the α1A/CMC column.