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Folate (vitamin B9) plays an important role in cell division and tissue growth by participating in the amino acid metabolism and nucleotide metabolism. Folate deficiency among pregnant women and children is common around the world, particularly in developing countries. Accurate folate analysis is critical for quality control in pharmaceutical and food products. This review briefly introduces the physiochemical properties, folate deficiency and food fortification of synthetic and natural folates, and the emphases are focused on the progress of sample pretreatment and analysis methods of these compounds in pharmaceuticals and foods in the last decade.