Milk, dialyzed milk and milk fat globule membrane (MFGM) (1.0g each) were boiled 3 min to inactivate pyrophosphatase. Release of flavins bound to milk proteins and deproteinization were performed simultaneously by Pronase digestion for 1 hr at 45°C. After centrifugation, the extracted flavins were analyzed by HPLC, using a C18 column at 40°C connected to a spectrofluorometer. Recovery of FAD, FMN and riboflavin was 97–99% from whole milk, 95–100% from dialyzed milk, and 99-100% from MFGM. The method is simple, rapid, sensitive and reproducible. Bovine milk contained 202 μg of total flavins per 100g milk, of which FAD was 13.8, FMN 4.3, and RF 81.9mol%(n=6).