The detection and assay of vitamin B-2 (Riboflavin) were conducted under aqueous conditions using various buffer solutions such as citrate buffer and sodium borate buffer at different pH levels. The absorbance spectrum of Riboflavin was measured across different pH values using these buffers. A spectrophotometer was utilized to achieve accurate and sensitive assay of Riboflavin at a wavelength of 440 nm. For preparation, a stock solution of Riboflavin with a concentration of 1.403 × 10−4 molar was prepared by dissolving the appropriate amount of the vitamin. Measurements included various aqueous solutions containing Riboflavin, such as aqueous test samples, vitamin capsules/tablets, and vitamin-water mixtures. UV/VIS spectrophotometry at 440 nm using different buffer solutions proved effective for assaying the B vitamin Riboflavin in aqueous media.