An enzyme-linked immunosorbent assay is reported for monitoring thevetin, an active constituent of the highly poisonous plant Thevetia nerifolia. A thevetin-BSA conjugate was employed as the immunogen and the antibodies raised in rabbits were used for the development of an ELISA. Penicillinase served as the marker enzyme and its conjugation to thevetin by the periodate method is reported for the first time. The present ELISA method could detect 2 ng/ml of thevetin. Cross-reactivity studies with structural analogues and other phytotoxins and drugs of common occurrence in clinical and forensic toxicology established the superiority of the ELISA over the existing analytical methods for determining thevetin in various biospecimens.