The effect of mobile phase pH, column temperature, extraction method and derivatisation time on the fluorescence response and recovery of fumonisin B1 in corn was investigated. Column temperature and mobile phase pH were negatively and positively correlated with the fluorescence response, respectively. Use of an Ultraturrax blender for extraction resulted in higher FB1 recoveries compared to a rotary shaker. In contrast to other reports, maximum fluorescence response occurred after a derivatisation time of 8 min. These results reflect on the absolute requirement for standardisation of the aforementioned parameters.