Abstract Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I‐III Regulatory, Quality, Manufacturing Postmarketing Phase IV Internal radioactive contamination can arise in different ways, from accidents affecting nuclear sites and industrial or medical sources, to the possible terrorist use of radiological dispersal devices. In all cases, internalized radionuclides may pose significant health risks and must be removed from the body. The need for safe, practical, and efficacious actinide chelation therapy options has motivated the current preclinical development of new hydroxypyridinone‐based actinide decorporation agents. In order to seek regulatory approval for such new decorporation agents, a number of efficacy studies must be performed and, in principle, must respond to selective criteria of the Animal Efficacy Rule from the US Food and Drug Administration (FDA). While many in vivo decorporation efficacy studies have been conducted over the past few decades using different animal species and contamination procedures, there is no designated standard animal model for the evaluation of new actinide chelation drugs. This study aims at probing the influence of a single variable such as the contamination dose on the efficacy of the chelators 3,4,3‐LI(1,2‐HOPO) and 5‐LIO(Me‐3,2‐HOPO) at removing plutonium‐238 administered as a soluble citrate complex. The dependence of body burden on contamination dose observed in untreated animals was reduced with the hydroxypyridinonate chelators, more so than with the commercially available diethylenetriaminepentaacetic acid chelator. This study demonstrates how a slight change in the design of decorporation experiments can remarkably impact the efficacy profile of the tested drug, and thus the path a sponsor might take to gain FDA approval.