Metabolic studies of polymethoxyflavones (PMFs) have become increasingly interesting to the scientific community. This is especially so in the areas of identification of in vivo biotransformation products because the elucidation and biological activity studies of PMFs metabolites are leading to further exploration of PMF's wide-ranging bioactivity. The metabolic pathway of tangeretin has been given a detailed examination, whereas the metabolic fate of other PMFs needs to more fully explored. Our in vivo transformation study of nobiletin in mice resulted in the finding of demethylated nobiletin metabolites isolated from urine and plasma. By comparing the Liquid Chromatography/Mass Spectrometry (LC/MS) results and Supercritical Fluid Chromatography (SFC) profiles of the synthesized hydroxylated PMFs with that of the metabolite mixture, three demethylated metabolites were identified in mouse plasma and urine as 3'-demethylnobiletin, 4'-demethylnobiletin and 3',4'-dihydroxy-5,6,7,8- tetra-methoxyflavone.