Plant physiological status is the interaction between the plant genome and the prevailing growth conditions. Accurate characterization of plant physiology is, therefore, fundamental to effective plant phenotyping studies; particularly those focused on identifying traits associated with improved yield, lower input requirements, and climate resilience. Here, we outline the approaches used to assess plant physiology and how these techniques of direct empirical observations of processes such as photosynthetic CO₂ assimilation, stomatal conductance, photosystem II electron transport, or the effectiveness of protective energy dissipation mechanisms are unsuited to high-throughput phenotyping applications. Novel optical sensors, remote/proximal sensing (multi- and hyperspectral reflectance, infrared thermography, sun-induced fluorescence), LiDAR, and automated analyses of below-ground development offer the possibility to infer plant physiological status and growth. However, there are limitations to such ‘indirect’ approaches to gauging plant physiology. These methodologies that are appropriate for the rapid high temporal screening of a number of crop varieties over a wide spatial scale do still require ‘calibration’ or ‘validation’ with direct empirical measurement of plant physiological status. The use of deep-learning and artificial intelligence approaches may enable the effective synthesis of large multivariate datasets to more accurately quantify physiological characters rapidly in high numbers of replicate plants. Advances in automated data collection and subsequent data processing represent an opportunity for plant phenotyping efforts to fully integrate fundamental physiological data into vital efforts to ensure food and agro-economic sustainability.