Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering, and determination of biomass composition is indispensable to accurate flux evaluation. We report the elemental and biomolecular composition of Catharanthus roseus hairy roots, a pharmaceutically significant plant system and an important metabolic engineering target. The molecular formula of the organic material in the hairy roots was C12.0H22.7N0.4O7.6 during mid-exponential growth. The abundances of lipids, lignin, cellulose, hemicellulose, starch, protein, proteinogenic amino acids, mineral ash, and moisture in the biomass were quantified. Analysis of water-soluble components of the biomass with 1-D C-13 and 2-D [ H-1, H-1] correlation ( COSY) NMR spectroscopy revealed that the water-soluble components were composed almost entirely of beta-glucans. Agropine, a frequently reported hairy root biomass component, was not detected. Our measurements of the biomass components quantified 83.6 ( 9.3% ( w/w) of the biomass. Together with previously reported abundances of indole alkaloids, we accounted for at least 85.9 ( 11.6% ( w/w) of the carbon in the biomass, which enabled the precise determination of 12 biomass synthesis fluxes.