Chlorophyll fluorescence is a noninvasive method to estimate the photosynthetic performance of organisms, from cyanobacteria to plants. Since the first mentions of chlorophyll a fluorescence in the literature, almost a century ago, it has been successfully used to gain insights into the performance and regulation of photosynthesis under different physiological and environmental conditions. This was possible due to the development of a theoretical framework combined with simple and affordable instrumentation. Several approaches have been designed, ranging across acquisition timescales (femtoseconds to seasons) and spatial dimensions, allowing the study of photosynthesis from a molecular perspective up to an ecosystem level. Nevertheless, their correct implementation and data interpretation require an understanding of the conceptual basis and models proposed. In this chapter, we will review the theoretical framework of chlorophyll fluorescence and discuss the most commonly used approaches and related parameters in a plant physiology context. We highlight the significance of chlorophyll fluorescence for understanding the photosynthetic regulation of plants under fluctuating environments.