Melanin is a critical pigment polymer in various organisms, essential for functions such as pigmentation and photoprotection. In humans, melanin primarily exists in three forms: eumelanin, pheomelanin, and neuromelanin. Eumelanin, responsible for dark skin and eye colors, is synthesized via tyrosinase-mediated pathways, converting L-tyrosine to L-DOPA and subsequently to DOPAchrome. Pheomelanin, associated with lighter skin and red hair, diverges from this pathway, incorporating cystine to produce yellow and red pigments. Neuromelanin, found in the brain, follows a similar initial pathway but diverges to form dopamine-derived polymers. Analytical techniques, particularly mass spectrometry, are crucial for understanding melanin’s structure and functions. Various ionization methods, including electrospray ionization and matrix-assisted laser desorption/ionization, have been employed to study melanin’s diverse forms and their roles in health and disease.