Several studies examining DNA deamination have published levels of 2′-deoxyinosine that illustrated a large variation between studies. Most of them are the result of artifactual DNA deamination that occurs during the process of sample preparation, particularly acid hydrolysis. This protocol for measurement of 2′-deoxyinosine describes the use of nuclease P1 and alkaline phosphatase to achieve release of nucleosides from DNA, followed by HPLC prepurification with subsequent gas chromatography–mass spectrometry analysis of the nucleosides. It has been used in the measurement of the levels of 2′-deoxyinosine in DNA of commercial sources and DNA from cells and animal tissues, and gives values ranging from 3 to 7 2′-deoxyinosine per 10⁶ 2-deoxyadenosine. This protocol should take approximately 7 days to complete.