Abstract Nanoparticle vehicles may improve the delivery of contrast agents and therapeutics to diseased tissues, but their rational design is currently impeded by a lack of robust technologies to characterize their in vivo behavior in real?time. This study demonstrates that fluorescent?labeled gold nanoparticles can be optimized for in vivo detection, perform pharmacokinetic analysis of nanoparticle designs, analyze tumor extravasation, and clearance kinetics in tumor?bearing animals. This optical imaging approach is non?invasive and high?throughput. Interestingly, these fluorescent gold nanoparticles can be used for multispectral imaging to compare several nanoparticle designs simultaneously within the same animal and eliminates the host?dependent variabilities across measured data. Together these results describe a novel platform for evaluating the performance of tumor?targeting nanoparticles, and provide new insights for the design of future nanotherapeutics.