A coupled in silico thermodynamic and probabilistic metabolic control analysis methodology was verified by applying it to the glycerol biosynthetic pathway in Saccharomyces cerevisiae. The methodology allows predictions even when detailed knowledge of the enzyme kinetics is lacking. In a metabolic steady state, we found that glycerol-3-phosphate dehydrogenase operates far from thermodynamic equilibrium ( -15.9 to -47.5 kJ mol(-1), where is the transformed Gibbs energy of the reaction). Glycerol-3-phosphataseoperates in modes near the thermodynamic equilibrium, far from the thermodynamic equilibrium or in between ( a parts per thousand 0 to -23.7 kJ mol(-1)). From the calculated distribution of the scaled flux control coefficients (median = 0.81), we inferred that the pathway flux is primarily controlled by glycerol-3-phosphate dehydrogenase. This prediction is consistent with previous findings, verifying the efficacy of the proposed methodology.