For immunization, saikosaponin a (SSa) was conjugated with bovine serum albumin (BSA). The hapten number in an antigen conjugate was determined to be eleven by matrix-assisted laser adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF Mass). Hybridomas secreting monoclonal antibodies (MAb) against SSa were produced by fusing splenocytes immunized with SSa-BSA conjugate and a hypoxanthine-aminopterin-thymidine-sensitive (HAT) mouse myeloma cell line, P3-X63-Ag8-653. A high specific MAb against SSa was selected from hybridomas using enzyme-linked immunosorbent assay (ELISA) analysis. Weak cross-reactivities occurred with saikosaponin c, b₂ and d, which are stereochemical and/or functional isomers of SSa, but no cross-reactivities were observed with other related steroidal glycosides. The full range of the assay extends 26 ng/ml to 1.5 μg/ml of SSa. Good correlation of SSa concentrations in a crude extract of Bupleuri radix between ELISA and HPLC methods was obtained after hydrolysis of acyl saikosaponins by treatment with a mild alkaline solution. The newly established ELISA has been applied for the quantitative assay of SSa in the Bupleuri radix and the Kampo medicines (TCM) prescribed with Bupleuri radix.