An efficient and convenient method, three-dimensional (3D) cell bioreactor coupled with HPLC/MS was developed for the screening and analysis of multiple bioactive components in herbal medicines.Cancer cells were cultured on a porous scaffold to form a 3D cell bioreactor.After interaction with live and fixed cancer cells, the HPLC fingerprinting chromatograms of herbal medicine extracts were compared to evaluate the binding properties of active components on cells.The well-known anticancer drugs resveratrol and paclitaxel were chosen to investigate the feasibility and this method was used to screen bioactive components from Polygonum Cillinerve (Nakai) Ohwi (PCO) extract.When the cell-drug interaction time was 30 min, the degrees of binding for 15 μg/ml of resveratrol and 15 μg/ml of paclitaxel were 66.1% and 82.2%, and those for the two main components in PCO extract (10 μg/ml), aristolochic acid A and aristolochic acid B, were 63.0% and 18.9% respectively.