In this study, an in vitro co-culture system of Castilleja tenuiflora and its host, Baccharis conferta, was used, and the impact of their interaction on specialized metabolite content was analyzed. After 4 weeks of co-culture, haustoria formation was verified through environmental scanning electron and confocal microscopy, confirming the successful establishment of the plant–plant interaction. Shoot height and biomass of the aerial part of the hemiparasite were not affected significantly by co-culture. However, root biomass increased by 53% compared to individually grown plants. Co-culture significantly reduced the host’s root length without negatively affecting its overall growth or survival. Phytochemical profile alterations were observed in both species. For C. tenuiflora, the lignans sesamin and eudesmin are proposed as differentially accumulated metabolites, while in B. conferta, the caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and the flavonoid acacetin were expressed differently. The development and chemical profiles of B. conferta and C. tenuiflora change when they grow in a co-culture because of the host–parasite interaction. Here, we report the feasibility of using a hemiparasite–host system to investigate more profound research questions. Future biotechnological applications of this system include elucidating the genetic regulators involved in haustorium formation, as well as optimizing environmental and physiological conditions to enhance its biosynthetic capacity for the production of specialized metabolites with therapeutic value.