Characterization of drug release under physiological conditions is very important for prediction of drug release in vivo, and can be used for formulation optimization. This assay uses a blood partitioning technique to measure the release of free drug from a nanoparticle-encapsulated drug complex. This assay is based on the principle that the free drug will partition between the plasma and red blood cell (RBC) fractions, whereas the hydrophilic nanoparticle platform is generally retained in the plasma fraction. The protocol is written for analysis of a dual radioactively labeled drug-nanoparticle platform complex (i.e. 14C-labeled drug and 3H-labeled nanoparticle platform). The protocol can also be adapted to use non-radioactive materials, provided that suitable analytical methods can be developed to measure both the drug and platform components independently in blood and plasma matrix (i.e. ICP-MS for the nanoparticle platform component and HPLC for the drug component). The same principles outlined in the study design below could be used for these non-radioactive analytical methods.