A method for determining urea in milk and milk powder was developed using high performance liquid chromatography with fluorescence detection (HPLC-FLD). Samples were extracted with a mixture of water and ethanol, the supernatant was derivatized with a solution of xanthydrol in acidic condition. The chromatographic separation was performed on a reversed-phase C18 column using a mobile phase consisting of acetonitrile and sodium acetate solution. The derivatized products were separated and tested using a fluorescence detector at λex 213 nm and λem 308 nm. The method showed a good linear relationship in the range of 0.1–10.0 μg/mL, with R2 > 0.999. The spiked recoveries of urea in formula milk powder and liquid milk samples were in the range of 104%–106% and 83.4%–101% with RSD 2.1% –5.7% and 3.2%–4.9%, respectively. The limits of detection (LODs) in formula milk powder and liquid milk were 30 and 12 mg/kg, respectively. This method proved rapid, sensitive, reliable and reproducible.