A semi-automated, metabolite prior-knowledge-based, lineshape fitting analysis has been developed to assess the dynamic biochemical changes found in ex vivo31P NMR pig liver preservation studies. Due to the inherent experimental limitations of the ex vivo study and the complexity of the composite phosphorus resonances, metabolite information obtained in vitro was incorporated into the ex vivo analysis. This approach has allowed complete metabolite analysis (phosphomonoesters, inorganic phosphate, phosphodiesters and nucleotide triphosphates) in over 2000 spectra in a fraction of the time compared with more conventional analysis methods. The developed analysis will enable complete and rapid assessment of the biochemical changes in ongoing cold preservation studies of the pig liver which will result in thousands of ex vivo31P NMR spectra. It is also envisaged that comparative studies on human donor livers will be carried out, in which this type of analysis would be the method of choice. Moreover, this kind of analysis approach could be advantageous in many complex in vivo NMR spectroscopy applications.