We present a magnetic particle imaging (MPI)-based assay for calculating the blood half-life and tissue uptake of magnetic nanoparticles (MNPs) without the need of labeling them. Dual-catheterized rats received 2.0 mg Fe of Synomag-D70, Synomag-D50, ferucarbotran, and Feraheme by femoral vein injection. The MPI signal of blood samples drawn from the femoral artery at various time points was then measured. Synomag-D70 exhibited biexponential clearance with half-lives of 3.2 and 31.2 min, Synomag-D50 a monoexponential clearance ( = 11.4 min), ferucarbotran a biexponential clearance ( = 2.4 and 10.8 min), and Feraheme a biexponential clearance ( = 60.9 and 4.5 min). MPI of perfused tissues showed MNPs primarily localizing in the spleen, liver, and lymph nodes. Spectrophotometric/chemical iron detection proved unreliable due to residual iron from endogenous blood. The MPI assay is a sensitive and specific method for evaluating the pharmacokinetics of existing MNP formulations and those in the pipeline, with exquisite sensitivity for ultrashort half-lives.