Tephritid fruit flies are highly successful invaders and some—such as the Mediterranean fruit fly, Ceratitis capitata (Wiedemann)—are able to adapt to a large range of crops. Biosecurity controls require that shipments of produce are ensured to be pest-free, which is increasingly difficult due to the ban of key pesticides. Instead, stress-based strategies including controlled atmosphere, temperature, and irradiation can be used to eradicate flies inside products. However, unlike pesticide science, we do not yet have a robust scientific approach to measure cost-effectively whether a sufficiently lethal stress has been delivered and understand what this stress does to the biology of the pest. The latter is crucial as it would enable a combination of stresses targeting multiple molecular pathways and thus allow for lower doses of each to achieve higher lethality and reduce the development of resistance. Using heat as an example, this is the first study investigating the molecular stress response to heat in Tephritidae. Using a novel setup delivering measured doses of heat on C. capitata larvae and a high-density 11 timepoint gene expression experiment, we identified key components of lethal heat-stress response. While unraveling the complete molecular mechanism of fruit fly response to lethal stress would be a long-term project, this work curates and develops 31 potential biomarkers to assess whether sufficient lethal stress has been delivered. Further, as these protocols are straightforward and less expensive than other—omic approaches, our studies and approach will assist other researchers working on stress response.