Betanin in solution, upon heating, was found to hydrolyze to betalamic acid and cyclodopa-5-O-glycoside. This reaction was monitored by an analytical high performance liquid chromatography (HPLC) method. The products were isolated by preparative reversed-phase HPLC or column chromatography using anion-exchange resins. Derivatives of betalamic acid (anilide, semicarbazone, condensation with L-proline) and cyclodopa-5-O-glycoside (hexacetate) were prepared as evidence to support the identification of these decomposition compounds. Formation of decarboxylated betanin was proposed based on the identification of CO2, chromatographic properties and the light absorption characteristics of the decarboxylated product.