<span class="paragraphSection"><div class="boxTitle">Abstract</div>Modifications on DNA and RNA, even at trace levels, play critical roles in diverse biological processes, yet their accurate quantification and discovery of novel modified constituents remain challenging. Here, we present a metabolic (deoxy)ribose-labeling approach integrated with multiple reaction monitoring mass spectrometry (MRM-MS) to enable sensitive and untargeted detection of nucleic acid modifications. In this approach, [U-¹³C]glucose is used to generate an ∼1:1 ratio of ¹³C₅-labeled and unlabeled deoxyribose moieties <span style="font-style: italic;">in vivo</span>, followed by MRM-MS acquisition of neutral-loss transitions corresponding to both isotopic forms. This isotopic pairing facilitates the differentiation of endogenous nucleosides from contaminants and allows confident assignment of authentic nucleoside signals. Applying this method to mouse embryonic stem cells, we detected rare nucleoside species such as 5-formylcytosine and 5-carboxycytosine, present at frequencies as low as one in 10⁶–10⁷ bases. In contrast, peaks assigned to <span style="font-style: italic;">N</span>⁶-methyladenine (6mA) lacked a labeled counterpart, suggesting that previously reported 6mA in mammalian DNA may arise from RNA misincorporation or artifacts introduced during the processing of isolated DNA. Analysis of formaldehyde-treated DNA revealed several previously unreported adducts, including <span style="font-style: italic;">N</span>⁴-hydroxymethyl-5-hydroxymethylcytosine (4hm5hmC, or dihmC). Collectively, this (deoxy)ribose-labeling strategy provides a robust and sensitive platform for untargeted nucleoside profiling and the discovery of unchara