A systematic investigation was carried out of the conditions employed for the post‐column iodination reaction used to enhance fluorescence sensitivity of aflatoxins B1 and G1 in reverse‐phase HPLC. The effect of post‐column reaction coil tubing dimensions, coil temperature, eluent and reagent flow rates and reagent concentration were examined and the optimum choice of each established. The maximum achievable sensitivity was shown to be 20 pg of B1 injected on column.