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We report validation of the first isotope dilution mass spectrometry method for determination of aflatoxin B1-N7-guanine (AFB1-N7-Gua), a major human aflatoxin−DNA adduct that is excreted in the urine. Measurement of urinary AFB1-N7-Gua, a biomarker of the biologically effective dose following dietary aflatoxin B1(AFB1) exposure, has helped identify AFB1as a risk factor in the development of hepatocellular carcinoma, a common cancer worldwide. Triple-quadrupole mass spectrometry, coupled with the use of a stable isotope-labeled internal standard (AFB1-N7-15N5-Gua) and better solid phase extraction and immunoaffinity column chromatography, have enabled us to greatly improve accuracy, precision, specificity, and sensitivity over previously published determinations. The limit of quantitation for AFB1-N7-Gua was 0.8 pg/20 mL urine (0.07 pg/mg creatinine). The method was validated for accuracy and precision over the range of 0.8−25 pg/20 mL urine, with between-day and within-day reproducibility for analysis of six aliquots of a human urine sample containing 6.0 pg/20 mL measured at <6% coefficient of variation. AFB1-N7-Gua concentrations were measured in 20 human urine samples collected in a region with known aflatoxin exposure. The mean concentration of AFB1-N7-Gua, measured in 16/20 urine samples with levels above the method's limit of quantitation, was 2.9 pg/20 mL urine (0.28 pg/mg creatinine) with a range of <0.8−7.2 pg/20 mL urine (0.04−65 pg/mg creatinine). With improved accuracy and precision, this sensitive biomarker for recent human exposure to AFB1will be especially useful for measuring the efficacy of planned interventions to reduce aflatoxin-related liver cancer in AFB1-exposed populations.