The front-face fluorescence spectroscopy technique was used to establish a rapid prediction model of riboflavin concentration in milk without prior sample preparation. The prediction model developed was then compared with two conventional high performance liquid chromatography (HPLC)-based quantification methods. The method of standard addition allowed detecting a linear correlation between fluorescence intensity and riboflavin concentration in 12% (w/w) reconstituted low-heat milk powder. Validation of the model yielded an R2 of 0.99 with a standard error of prediction of 0.13 mg/L. The results suggest a potential use of front-face fluorescence spectroscopy as a simple method for off- and in-line determinations of riboflavin in milk.