The recent introduction of multiphoton microscopy coupled with advances in optics, computer sciences, designer fluorophores, molecular labeling, and previously developed physiologic approaches have empowered investigators to quantitatively study the cell-specific dynamic events, such as endocytosis, within a functioning organ with subcellular resolution. This rapidly emerging field of investigation, with superior spatial and temporal resolution and high sensitivity, enables investigators to track molecules and determine their mode of cellular uptake, intracellular trafficking, and metabolism in a cell-specific fashion in complex heterogeneous organs such as the kidney with repeated determinations possible over a prolonged period of time. This approach is enhanced by the ability to obtain and quantify volumetric data with using up to three different fluorophores simultaneously. We have utilized this intravital approach to understand and quantify kidney proximal tubule cell uptake and intracellular distribution and metabolism of fluorescently labeled molecules, including folic acid, gentamicin, and small interfering ribonucleic acid (siRNA). Limitations of this technique include tissue penetration, which is the major barrier to successful clinical utilization of this technology. However, its use in preclinical animal models offers new insight into physiologic processes and the pathophysiology and treatment of disease processes.