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Introduction: The biological consequences of absorbed radiation doses are ill-defined for radiopharmaceuticals, unlike for external beam radiotherapy (EBRT). A reliable assay that assesses the biological consequences of any radionuclide is much needed. Here, we evaluated the cell-free plasmid DNA assay to determine the relative biological effects of radionuclides such as Auger electron-emitting [ 67 Ga]GaCl 3 or [ 111 In]InCl 3 compared to EBRT., Methods: Supercoiled pBR322 plasmid DNA (1.25 or 5 ng/μL) was incubated with 0.5 or 1 MBq [ 67 Ga]GaCl 3 or [ 111 In]InCl 3 for up to 73 h or was exposed to EBRT ( 137 Cs; 5 Gy/min; 0-40 Gy). The induction of relaxed and linear plasmid DNA, representing single and double strand breaks, respectively, was assessed by gel electrophoresis. Chelated forms of 67 Ga were also investigated using DOTA and THP. Topological conversion rates for supercoiled-to-relaxed (k sr x ) or relaxed-to-linear (k rl x ) DNA were obtained by fitting a kinetic model., Results: DNA damage increased both with EBRT dose and incubation time for [ 67 Ga]GaCl 3 and [ 111 In]InCl 3 . Damage caused by [ 67 Ga]GaCl 3 decreased when chelated. [ 67 Ga]GaCl 3 proved more damaging than [ 111 In]InCl 3 ; 1.25 ng/μL DNA incubated with 0.5 MBq [ 67 Ga]GaCl 3 for 2 h led to a 70% decrease of intact plasmid DNA as opposed to only a 19% decrease for [ 111 In]InCl 3 . For both EBRT and radionuclides, conversion rates were slower for 5 ng/μL than 1.25 ng/μL plasmid DNA. DNA damage caused by 1 Gy EBRT was the equivalent to damage caused by 0.5 MBq unchelated [ 67 Ga]GaCl 3 and [ 111 In]InCl 3 after 2.05 ± 0.36 and 9.3 ± 0.77 h of incubation, respectively., Conclusions: This work has highlighted the power of the plasmid DNA assay for a rapid determination of the relative biological effects of radionuclides compared to external beam radiotherapy. It is envisaged this approach will enable the systematic assessment of imaging and therapeutic radionuclides, including Auger electron-emitters, to further inform radiopharmaceutical design and application., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)