Thapsigargin and related compounds are produced by Thapsia garganica L. (Apiaceae) and are thought to be a defence compound against herbivory. Thapsigargin inhibits the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) in both vertebrates and invertebrates. This activity is responsible for its potent toxicity, as well as the potential use to treat solid tumours. However, the ecological role and regulation of thapsigargin are not well understood, and the aim of this study was to investigate if thapsigargin biosynthesis was responsive to leaf damage. To test the response to potential leaf damage during a herbivory, greenhouse plants were subjected to clipping to mimic the physical damage. Unclipped versus clipped plants were sampled for chemical analysis and the gene expression for the two known thapsigargin biosynthetic genes (TgTPS2 and TgCYP76AE2) was investigated. Data obtained by LC–ESI–MS/MS were used to perform molecular networking to identify chemical constituents related to thapsigargin and its biosynthesis. The results show a significant change in a plant’s chemical profile after mimicking an herbivory event. Both the chemical analysis and gene expression data show that T. garganica plants can induce the biosynthesis of this class of defence compounds at the site of an attack. Thapsigargins are clearly the dominant defence compounds in these plants, and they seem to be produced through a common biosynthetic pathway with little diversity. This likely means that T. garganica has a relatively simple response to herbivory, as opposed to many other plant species that have been shown to have complex metabolite responses to herbivory.