The amount of 5-methyl-2'-deoxycytidine (m5dC) and its oxidized derivatives 5-hydroxymethyl-dC (hm5dC), 5-formyl-dC (f5dC), and 5-carboxy-dC (ca5dC) inside mammalian cells provides valuable information concerning cellular state and fate. LC-MS methods enable reliable quantification of these noncanonical DNA modifications in the low femtomolar range. Here, we describe a broadly applicable protocol to quantify m5dC, hm5dC, f5dC, and ca5dC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).