8-Oxo-7,8-dihydro-2′-deoxyguanosine (dOG), a well-studied oxidation product of 2′-deoxyguanosine (dG), is prone to facile further oxidation forming spiroiminodihydantoin 2′-deoxyribonucleoside (dSp) in the nucleotide pool and in single-stranded oligodeoxynucleotides (ODNs). Many methods for quantification of damaged lesions in the genome rely on digestion of DNA with exonucleases or endonucleases and dephosphorylation followed by LC-MS analysis of the resulting nucleosides. In this study, enzymatic hydrolysis of dSp-containing ODNs was investigated with snake venom phosphodiesterase (SVPD), spleen phosphodiesterase (SPD) and nuclease P1. SVPD led to formation of a dinucleotide, 5′-d(Np[Sp])-3′ (N = any nucleotide) that included the undamaged nucleotide on the 5′ side of dSp as the final product. This dinucleotide was a substrate for both SPD and nuclease P1. A kinetic study of the activity of SPD and nuclease P1 showed a sequence dependence on the nucleotide 5′ to the lesion with rates in the order dG > dA > dT > dC. In addition, the two diastereomers of dSp underwent digestion at significantly different rates with dSp1 > dSp2; nuclease P1 hydrolyzed the 5′-d(Np[Sp1])-3′ dinucleotide two- to six-fold faster than the corresponding 5′-d(Np[Sp2])-3′, while for SPD the difference was two-fold. These rates are chemically reasoned based on dSp diastereomer differences in the syn vs. anti glycosidic bond orientation. A method for the complete digestion of dSp-containing ODNs is also outlined based on treatment with nuclease P1 and SVPD. These findings have significant impact on the development of methods to detect dSp levels in cellular DNA.