Flavin adenine dinucleotide (FAD), a versatile metabolic cofactor, is emerging as an important non-canonical RNA cap across various life domains. This review explores FAD's dual role as a coenzyme and an RNA modifier, focusing on its incorporation as a 5' cap structure during transcription initiation and its subsequent implications for RNA metabolism and cellular functions. A comprehensive view of the mechanisms underlying FAD capping and decapping is presented, highlighting key enzymes that play a role in these processes. FAD-capped RNA is shown to play critical roles in viral replication, as demonstrated in the Hepatitis C virus, where FAD capping supports cellular immune evasion. Analytical techniques, including mass spectrometry and innovative sequencing methodologies, have advanced our understanding of the flavin cap, enabling its identification and quantification in different biological systems. This review underscores the significance of FAD-RNA capping as a novel regulatory mechanism, proposes innovative methodologies for its study, and emphasizes its potential therapeutic applications in viral and cellular biology.