Aflatoxins (AF) are group I human carcinogen and potent mycotoxins that commonly contaminate peanuts, corns, and their products during growth, harvest and storage. Exposure to AF is linked to growth stunting in children, which has been extensively studied in recent years. With increasing involvement of young children as study subjects, dried blood spot (DBS) rose in popularity as an important alternative blood collection strategy. In this study, an HPLC-fluorescence method measuring exposure biomarker of AFB1, AFB1-lysine adduct, was developed and validated. For initial method development and validation, DBS and serum samples were obtained from AFB1-treated F344 rats, as well as selected samples from human field study, which were analyzed and compared, and has found good correlation between serum and DBS samples (0.997 for the single-dose exposure and 0.996 for repeated dose exposure). DBS samples (n=393) from Nepal were analyzed and compared with the results from matching serum samples. The result suggested that the HPLC-florescence method is effective in detecting AFB1-lysine adducts and can potentially be applied in epidemiological studies.