A four-part method based on Wahba's technique is presented for the specific determination of the riboflavin content of children's urine. First, the interfering urinary constituents are precipitated by treating the urine sample with zinc acetate and formalin solutions; they are then removed by centrifugation. The riboflavin in the resultant supernatant liquid is next separated from the other soluble molecular constituents of urine by elution chromatography, by using a talc column. The riboflavin eluate is then subjected to one-dimensional chromatography on silica gel plates to confirm the identity of the fluorescent spot before the determination. Finally, the thin-layer chromatographic plate is dried and the riboflavin content of the plate is measured fluorimetrically by using the technique of reflectance densitometry.