DNA alkylating drugs have been used as cancer chemotherapy with variable outcomes. The establishment of predictive biomarkers to identify patients who will effectively respond to treatment would allow for the development of personalized therapies. As the degree of interaction of alkylating drug with DNA plays a key role in their mechanism of action, our hypothesis is that the measurement of the DNA adducts formed by alkylating drugs could be used to inform patient stratification. Beginning with busulfan, we took advantage of our DNA adductomic approach to characterize DNA adducts formed by reacting busulfan with calf-thymus DNA. Samples collected from six patients undergoing busulfan-based chemotherapy prior to allogeneic hematopoietic cell transplantation were analyzed for the presence of busulfan-derived DNA adducts. Among the 15 adducts detected in vitro, 12 were observed in the patient blood confirming the presence of a large profile of DNA adducts in vivo. Two of the detected adducts were structurally confirmed by comparison with synthetic standards and quantified in patients. These data confirm our ability to comprehensively characterize busulfan-derived DNA damage and set the stage for the development of methods to support personalized chemotherapy.