Fumonisins (FBs), mycotoxins specially found in maize, interfere with sphingolipid metabolism, altering the sphinganine (Sa) to sphingosine (So) ratio. In humans, FBs have been epidemiologically associated with esophageal cancer. The exposure evaluation of different populations is of primordial interest for public health maintenance. This might be accomplished through the determination of biomarkers, such as the Sa/So ratio, in biological samples. This paper presents the optimization of an analytical methodology for the determination of the Sa/So ratio in human urine. Isolation of exfoliated cells from urine, followed by extraction with ethyl acetate, derivatisation with naphthalene-2,3-dicarboxaldehyde (NDA), and detection and quantification by liquid chromatography (LC) with fluorescence detection (FD) allowed adequate sensitivity, accuracy, and precision. The application of this analytical procedure to a rural and an urban population from the Central zone of Portugal demonstrated that the Sa/So ratio was 0.43+/-0.22 and 0.42+/-0.17, respectively. Regarding the rural population, the Sa/So ratio presented mean levels of 0.48+/-0.22 for females, and 0.30+/-0.16 for males. Concerning the urban population, the Sa/So ratio presented a mean of 0.44+/-0.18 for females, and was 0.29 in the only male sample where its establishment was possible. The statistical analysis showed that no significant differences were found between the different studied populations.