The clinical sequela of mitochondrial toxicity in the proximal tubule (PT) depends on the severity and ranges from asymptomatic increases in urine low molecular weight protein (LMWP) excretion to significant wasting of solutes. Fluorescence microscopy is the only method available that allows simultaneous combined assessment of mitochondrial morphology, dynamics, and function in situ in living cells and therefore represents a powerful tool in mitochondrial research. Several important mitochondrial signals can be imaged in intact kidney tissue, both ex vivo and in vivo, using endogenous and exogenous fluorescent molecules. Using intravital multiphoton microscopy (MPM), this chapter investigates how disease-causing insults affect mitochondrial function in the kidney. The chapter also investigates how mitochondria in the kidney respond to therapeutic drugs that cause nephrotoxicity. It shows that live imaging of mitochondria can be performed in both kidney slices and the isolated perfused kidney (IPK) and that there are striking differences in intrinsic mitochondrial signals between different nephron segments.