Time-resolved fluorescence spectroscopy (TRFS) is a powerful, noninvasive technique for probing ultrafast energy transfer, structural organization, and charge separation processes within photosynthetic machinery in vivo. Furthermore, TRFS measurements on intact leaves provide valuable insights into the response of the photosynthetic machinery to light stress or other abiotic changes. TRFS enables the monitoring of the primary steps of photosynthesis on picosecond to nanosecond timescales. Despite its widespread application for isolated complexes and unicellular organisms, the use of TRFS on intact leaves presents unique technical challenges due to the complexity of the sample. These challenges include minimizing excitation-induced annihilation effects, ensuring well-defined photosynthetic states, maintaining leaf integrity, and accounting for spectroscopic artifacts caused by tissue heterogeneity, scattering, and light reabsorption. Therefore, proper experimental considerations and configurations are required to mitigate these factors and ensure reliable and interpretable fluorescence decay measurements. TRFS thus remains an essential tool for advancing the understanding of photosynthetic performance in plants, plant stress responses, and strategies for improving crop productivity. This chapter reviews the principles, methodologies, and challenges associated with time-resolved fluorescence measurements in intact leaves, highlighting current advancements and knowledge gaps in the field.