The Madagascar periwinkle (Catharanthus roseus) produces over 100 alkaloids, many of which show activity against cancers and other diseases. These specialized metabolites are one very important way that the plant protects itself from predators, microbes, and other competitors. In this study, we investigate the alkaloid profile of in vitro tissue cultures that have been elicited with a plant defense hormone in order to increase and diversify biosynthesis. Their alkaloid profiles, including at least one previously uncharacterized metabolite, are compared and characterized by MS/MS. A comparison of the alkaloid profiles from C. roseus native roots, untreated, and elicited hairy root lines suggests that elicitation with the plant defense hormone, methyl jasmonte, uniquely altered alkaloid biosynthesis by diverting the metabolic pathway. Building on this concept, it is extrapolated that by creating tissue cultures of other plants of medicinal interest, we may be able to efficiently and reliably create unique accumulations of metabolites that the plant produces as a specific and reproducible defense response, therefore increasing the chance for the production of bioactive defense compounds.