Riboflavin (vitamin B2) is an essential vitamin which is utilized in a variety of biological functions. Intracellularly riboflavin is phosphorylated to riboflavin 5′phospate (FMN) or converted to flavin adenine dinucleotide (FAD) to play crucial coenzyme roles in the respiration pathways. Deficiencies in this vitamin (known as ariboflavinosis) generally lead to fatigue, skin disorders, and hyperemia. The following chapter describes a simple and reproducible method for the analysis of riboflavin via high-performance liquid chromatography utilizing fluorescence detection.