Immunoaffinity column-based sample preparation procedures for determination of aflatoxins B1, B2, G1, and G2 in several food matrixes and aflatoxin M1 in milk have been automated by using flexible automation, or robotics. Components used to assemble the system were purchased commercially or developed and built in-house. A liquid-level sensor developed in-house to assist elution of the immunoaffinity column is described. After immunoaffinity column cleanup, aflatoxins are separated by reversed-phase liquid chromatography and determined by fluorescence without derivatization. Mean recoveries of aflatoxins B1, B2, and G1 added to corn and nuts at 9-36 ng/g total aflatoxins were > 85% (coefficient of variation [CV] = 16%). Recoveries of aflatoxin G2 averaged 50% (CV = 28%). Recoveries of aflatoxin M1 added to milk at 0.12-0.50 ng/mL averaged 78% (CV = 19%). The ability of the automated system to reproduce its results is demonstrated by the fact that the CV of replicate assays is generally better than 10%. Comparability between the automated procedure and the AOAC official method is demonstrated.