Technical Abstract: Cyclopamine was isolated from Veratrum californicum and identified as the teratogen responsible for severe craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in central Idaho. More recently, cyclopamine was found to inhibit the hedgehog (Hh) signaling pathway which plays a critical role in embryonic development and is implicated in several types of cancer. Thus, cyclopamine and cyclopamine derivatives have been targeted as potential pharmaceutical treatments for certain cancers and other diseases associated with the Hh signaling pathway. A monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay was developed to detect and measure cyclopamine and cyclopamine derivatives in biological samples. The limits of detection of the assay for cyclopamine, cyclopamine-KAAD, and compound (11) were 2.9 pg, 0.41 pg and 2.6 pg, respectively. This assay was also found to be useful for the detection and measurement of cyclopamine in sera from mice that had been injected with cyclopamine. The simple ELISA method described herein demonstrates the potential of using these techniques for the rapid screening of biological samples for the presence and levels of cyclopamine and other cyclopamine derivatives that are Hh inhibitors with anticancer potential.