We have developed a noninvasive imaging method to quantify in vivo drug delivery pharmacokinetics without the need for blood or tissue collection to determine drug concentration. By combining the techniques of hyperspectral imaging and a dorsal skinfold window chamber, this method enabled the real-time monitoring of vascular transport and tissue deposition of nanoparticles labeled with near-infrared (NIR) dye. Using this imaging method, we quantified the delivery pharmacokinetics of the native high-density lipoprotein (HDL) and epidermal growth factor receptor (EGFR)-targeted HDL nanoparticles and demonstrated these HDLs had long circulation time in blood stream (half-life >12 h). These HDL nanoparticles could efficiently carry cargo DiR-BOA to extravasate from blood vessels, diffuse through extracellular matrix, and penetrate and be retained in the tumor site. The EGFR targeting specificity of EGFR-targeted HDL (EGFR-specific peptide conjugated HDL) was also visualized in vivo by competitive inhibition with excess EGFR-specific peptide. In summary, this imaging technology may help point the way toward the development of novel imaging-based pharmacokinetic assays for preclinical drugs and evaluation of drug delivery efficiency, providing a dynamic window into the development and application of novel drug delivery systems.