<p>Conventional inductively coupled plasma mass spectrometry (ICP-MS) merely measures total phosphorus within nucleic acid matrices and cannot resolve intact target DNA from degraded oligonucleotide fragments or matrix-originated phosphorus contaminants. In this work, we report a hyphenated size-exclusion chromatography-ICP-MS (SEC-ICP-MS) workflow for direct mass quantification of double-stranded DNA fragments spanning hundreds to thousands of base pairs. Purified DNA fragments are introduced directly via HPLC injection for size-based separation. ³¹P¹⁶O signals and ¹¹⁵In signals are simultaneously monitored by ICP-MS, with indium serving as the internal standard. External calibration using certified inorganic phosphorus solution enables phosphorus quantification, and DNA mass concentrations are subsequently derived from the fixed stoichiometric phosphorus fraction of each defined DNA sequence. The instrumental limit of detection for phosphorus reaches 28&#xa0;ppb, corresponding to an absolute mass limit of 112&#xa0;pg. Method trueness was validated with two independent nucleic acid certified reference materials, with relative deviations falling between 1.2 and 5.0%, verifying favorable quantitative accuracy. The workflow was applied to two synthetic DNA fragments (300&#xa0;bp and 2000&#xa0;bp), giving average mass concentrations of 141.8 ± 3.7&#xa0;mg·kg⁻¹ and 110.5 ± 3.1&#xa0;mg·kg⁻¹, respectively. Orthogonal cross-validation via the&#xa0;digital polymerase chain reaction (dPCR) produced consistent measurements of 140.5 ± 5.1&#xa0;mg·kg⁻¹ (300&#xa0;bp) and 113.3 ± 4.3&#xa0;mg·kg⁻¹ (2000&#xa0;bp), jointly corroborating the robust reliability of this fragment-resolved SEC-ICP-MS quantification strategy. The developed SEC-ICP-MS workflow delivers robust, fragment-resolved absolute quantification for short-to-long DNA constructs and exhibits compatibility with functionalized modified nucleic acids.</p> Graphical abstract <p></p>