Inorganic phosphate (Pi) has central roles in metabolism, cell signaling, and energy conversions that rely on the distribution of appropriate amounts of Pi to each cell and subcellular compartment. An analytical method for monitoring Pi dynamics with high spatial and temporal resolution is therefore required to gain a comprehensive understanding of the transport and metabolic recycling mechanisms that govern Pi homeostasis. In this work I optimized a genetically encoded Forster Resonance Energy Transfer (FRET)-based Pi biosensor to assess cellular and subcellular Pi concentrations. Using a model animal, Caenorhabditis elegans, I demonstrated that the Pi biosensor could resolve cell-specific and developmental stage-specific differences in cytosolic Pi accumulation. This study also established that cellular Pi concentration is a sensitive indicator of metabolic status. I further refined the methods of FRET-based Pi measurements for different subcellular compartments in the model plant Arabidopsis thaliana. Additionally, I used microinjection to develop an in vivo calibration for Pi-dependent FRET responses that enabled direct quantification of Pi within the cytosol. Using this method, I identified an unexpected developmental zone-specific Pi concentration pattern in the root, with highest accumulation in the transition zone. This study revealed that the Pi distribution pattern is robust, independent of external Pi, and is modulated by intracellular activities. Finally, I used FRET-based Pi measurements to establish that the chloroplast Pi transporter PHT2;1 imports Pi into the chloroplast and thereby modulates stromal Pi concentrations.