Geniposide, Geniposide, the main active component in extracts of Gardenia jasminoides ELLIS., is one of the main components of Huanglian-Jiedu-Tang (HJT). This study aimed to validate an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on monoclonal antibodies (mAb) against geniposide (anti-geniposide mAb), which was developed by our lab, and apply the assay to study the pharmacokinetics of geniposide in HJT in mice. Blood samples were drawn from mice at predetermined time points after oral administration of HJT in three dosages. A linear correlation was obtained for geniposide concentrations in the range from 1.17 to 37.50 µg/mL. The intra-day and inter-day precision values of the icELISA method were well within the recommended range (≤10%). The recovery rates ranged from 99.74 to 102.40%. Stability studies showed that geniposide sample solutions were intact for 12 h. The Tmax and mean residence time (MRT) of geniposide of the three groups were consistent with previous data. The results suggest that a reliable and effective method was established and could be applied to the study of the pharmacokinetics of geniposide in HJT.