To date, few studies have evaluated sub-organismal responses (e.g., physiological or energetic consequences) of individual fish to hydropower infrastructure (e.g., fishways, turbines) or operations (e.g., fluctuating flows, low flows). The field of “conservation physiology” (i.e., the use of physiological information to enhance conservation) is expanding rapidly and has great promise for hydropower research. However, there is a need to both expand the “toolbox” available to practitioners and to validate these tools for use in this context. This synthetic report details the behavioural, energetic, genomic, molecular, forensic, isotopic, and physiological tools available for studying sub-organismal responses of fish to hydropower infrastructure and operating procedures with a critical assessment of their benefits and limitations. Furthermore, this paper provides two case studies where behavioural, energetic, and physiological tools have been used in hydropower settings. Progressive and interdisciplinary approaches to hydropower research are needed to advance the science of sustainable river regulation and hydropower development. The expanded toolbox could be used by practitioners to assess fishway performance, migration delays, and fish responses to fluctuating flows through a more mechanistic approach than can be offered by only focusing on population metrics or indices of community structure. These tools are also relevant for the evaluation of other anthropogenic impacts such as water withdrawal for irrigation or drinking water, habitat alteration, and fisheries interactions.