Technical Abstract: Veratrum californicum was responsible for large losses in sheep grazing high mountain ranges in central Idaho in the 1950's. Veratrum induces various birth defects including the cyclopic-type craniofacial defect (monkey faced lambs) that is specifically induced in lambs after pregnant ewes grazed the plant on the 14th day of gestation. The steroidal alkaloids, cyclopamine and jervine, were isolated from Veratrum and shown to be primarily responsible for the malformations. Cyclopamine and jervine are potent teratogens that inhibit Sonic hedgehog (Shh) signaling during gastrulation stage embryonic development, producing cyclopia and holoprosencephaly. While losses to the sheep industry from Veratrum are now relatively infrequent, occasional incidents of toxicoses and craniofacial malformations are still reported in sheep an other species. However, the benefits to biomedical research using cyclopamine as a tool to study human diseases have greatly expanded. A competitive inhibition enzyme-linked immunosorbent assay to detect and measure cyclopamine and jervine was developed using polyclonal antibodies. The limits of detection of the assay were 90.0 pg and 22.7 pg for cyclopamine and jervine, respectively. This assay was used for the detection and measurement of cyclopamine spiked into sheep blood. The simple extraction-ELISA methods developed in this study demonstrate the potential of using these techniques for the rapid screening of biological samples to detect the presence and concentration of cyclopamine and jervine and will be beneficial to pharmacological studies and livestock diagnostics.