Enzyme immunoassay of glycyrrhetic acid was developed by employing antisera elicited from in rabbits immunized with C-3 bridged glycyrrhetic acid.sbd.bovine serum albumin (BSA) conjugate (15, 16) and C-3 bridged glycyrrhetic acid.sbd..beta.-galactosidase conjugate (17, 18) as labeled antigens. Hemisuccinyl and hemiglutaryl groups, which were chosen as the chemical bridges, were introduced at the hydroxy group of tert-butyl glycyrrhetate (2). Immunoassay was performed with bridge heterologous combinations such as anti-glycyrrhetic acid-hemisuccinate-BSA (15) antiserum and glycyrrhetic acid.sbd.hemiglutarate.sbd..beta.-galactosidase conjugate (18) and the reverse combination (16 and 17). The sensitivity of enzyme immunoassays (EIA) expressed as the midpoint (logit B/B0 = 0) was higher in the former combination (2.4 ng/tube) than the reverse combination (8.6 ng/tube). The cross reactivities of the anti-C-3 bridged glycyrrhetic acid antisera with C-3 substituted derivatives of glycyrrhetic acid were higher than those of the anti-C-30 bridged glycyrrhetic acid antiserum.