BACKGROUND Aflatoxins (AFs) are carcinogenic mycotoxins. A simple, quick, and accurate method for the micro-analysis of AFs in foodstuffs, especially spices, is needed. OBJECTIVE A sophisticated pretreatment method that combines solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization using immunoaffinity (IA) gel as the solid phase was developed to analyze AFs in spices simply, quickly, and sensitively by liquid chromatography with fluorescence detection. METHODS White and black pepper samples were extracted with a mixed solution of methanol/water (4:1) and then diluted with 7% aqueous solution of Triton-X. The solution was subjected to cleanup by SPDE using IA gel. Trifluoroacetic acid was added to the IA gel for on-site solid-phase fluorescence derivatization. RESULTS Chromatograms containing well-separated peaks and few interference peaks from contaminants were obtained. The method detection limit of AFs in white and black pepper was 0.15-0.29 ng/g. Repeatability and intermediate precision were <10% and <15%, respectively, and accuracy was 61.7-87.8%. In addition, inter-laboratory precision was <29% and mean recovery was 61.5-76.7%. A favorable z-score of |Z| ≦ 1 was obtained in seven laboratories, although one laboratory gave 2 < |Z| < 3. CONCLUSIONS The validity, reliability, practicality, and robustness of the developed method were verified. CONCLUSIONS By using SPDE and solid-phase fluorescence derivatization in combination for AF analysis, fluorescence derivatization during cleanup was realized, leading to simplification of the pretreatment operation.