A method has been developed to determine the presence of aflatoxin B1 in the urine of animals (including humans) by utilizing commercial immunochemical kits that can be used in the field. Urine is treated with diatomaceous earth and filtered to clarify the sample; 2-3 ppb aflatoxin B1, corresponding to about 300 ppb in the ingested feed/food, can be detected in the filtered urine without further purification. To improve sensitivity, the urine filtrate is passed through a C18 solid phase column to extract the aflatoxin. The column is washed with acetonitrile-water (15 + 85) and water, aflatoxin B1 is eluted with methanol-water (7 + 3), and water is added to the eluate, which is then tested for aflatoxin with the test kit. The limit of detection is 0.2 ppb, reflecting consumption of 40 ppb or more aflatoxin in the feed/food. When the initial sample volume is adequate, purification through the C18 column step is usually sufficient. For limited sample volumes, the eluate from the C18 column is mixed with water, added to an immunosorbent affinity column, and washed with water to remove excess sample matrix and impurities. Aflatoxin B1 is eluted with acetonitrile. The extract is evaporated under nitrogen and the residue is redissolved in methanol-water (25 + 75). At this purification stage, the limit of detection is reduced to 0.05 ppb.