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.