In a previous study, we reported the preparation, characterization, variation, specificity, and sensitivity of an anti-aristolochic acid-II (AA-II) monoclonal antibody. The preparation procedure was as follows. AA-II conjugated with bovine serum albumin was used as an antigen for immunizing BALB/c mice. Splenocytes isolated from the immunized mice were fused with an aminopterin-sensitive mouse myeloma cell line to produce hybridoma cells secreting a mono-clonal antibody (MAb) against AA-II. The selected MAb was subsequently cloned. Hapten number, isotype, and an esti-mated dissociation constant (KD) of the secreted MAb were determined. This MAb was used to establish an ELISA method. The linear range was 0.19-13 µg/ml. Anti-AA-II MAb showed extremely high specificity for AA-II, low cross-reactivity (CR) against other AAs or aristololactam-I, and negligible CR (<0.5%) toward other natural compounds with different chemical structures. This study describes the successful application of the ELISA method using anti-AA-II MAb to determine AA-II concentration in several crude drugs derived from Aristolochia species. The highest AA-II concentration (2.82 µg/mg) was observed in the stem of A. manshuriensis, followed by that in the fruit of A. contorta (0.81 µg/mg). In case of A. indica, AA-II concentration in the root was higher than that in the aerial parts. These data indicated that the established ELISA method can be used for the quality control of crude drugs derived from Aristolochia plants.