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Humans are unavoidably exposed to numerous different mutagenic DNA alkylating agents (AAs), but their role in the initiation of cancers is uncertain, in part due to difficulties in assessing human exposure. To address this, we have developed a screening method that measures promutagenic <i>O</i>⁶-alkylguanines (<i>O</i>⁶-AlkGs) in DNA and applied it to human DNA samples. The method exploits the ability of the <i>Schizosaccharomyces pombe</i> alkyltransferase-like protein (Atl1) to recognise and bind to a wide range of <i>O</i>⁶-AlkGs in DNA. We established an Atl1-based slot-blot (ASB) assay and validated it using calf thymus DNA alkylated in vitro with a range of alkylating agents and both calf thymus and human placental DNA methylated in vitro with temozolomide (TMZ). ASB signals were directly proportional to the levels of <i>O</i>⁶-meG in these controls. Pre-treatment of DNA with the DNA repair protein <i>O</i>⁶-methylguanine–DNA methyltransferase (MGMT) reduced binding of Atl1, confirming its specificity. In addition, MCF 10A cells were treated with 500 μM TMZ and the extracted DNA, analysed using the ASB, was found to contain 1.34 fmoles <i>O</i>⁶ -meG/μg DNA. Of six human breast tumour DNA samples assessed, five had detectable <i>O</i>⁶-AlkG levels (mean ± SD 1.24 ± 0.25 <i>O</i>⁶-meG equivalents/μg DNA. This study shows the potential usefulness of the ASB assay to detect and quantify total <i>O</i>⁶-AlkGs in human DNA samples.