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Nucleic acid nanoparticles (NANPs) are increasingly used in preclinical investigations as delivery vectors. Tools that can characterize assembly and assess quality will accelerate their development and clinical translation. Standard techniques used to characterize NANPs, like gel electrophoresis, lack the resolution for precise characterization. Here, we introduce the use of charge detection mass spectrometry (CD-MS) to characterize these materials. Using this technique, we determined the mass of NANPs varying in size, shape, and molecular mass, NANPs varying in production quality due to formulations lacking component oligonucleotides, and NANPs functionalized with protein and nucleic acid-based secondary molecules. Based on these demonstrations, CD-MS is a promising tool to precisely characterize NANPs, enabling more precise assessments of the manufacturing and processing of these materials.