A method for metabolic flux determination by carbon labeling experiments is described. The cells were grown on C-13-labeled substrate, and the labeling patterns of the amino acids in biomass hydrolysate were measured using two dimensional H-1-C-13 correlation nuclear magnetic resonance spectroscopy (2D H-1-C-13 COSY NMR) and gas chromatography/mass spectrometry (GC-MS) technique. The estimation of intracellular flux distribution from the measured labeling data and extracellular fluxes was achieved by a nonlinear least squares fitting approach, since a framework was developed that allows mathematical description relating the measured quantities and the intracellular fluxes. The described method was used to investigate the central carbon metabolism of the mixotrophically cultivated Synechocystis sp. PCC6803. The statistical quality of the estimated fluxes was also evaluated.