Aim: The purpose of this research was to use metabolomics to investigate the cystic phenotype in the Lewis polycystic kidney rat. Methods: Spot urine samples were collected from four male Lewis control and five male Lewis polycystic kidney rats aged 5 weeks, before kidney function was significantly impaired. Metabolites were extracted from urine and analysed using gas chromatography-mass spectrometry. Principal component analysis was used to determine key metabolites contributing to the variance observed between sample groups. Results: With the development of a metabolomics method to analyse Lewis and Lewis polycystic kidney rat urine, 2-ketoglutaric acid, allantoin, uric acid and hippuric acid were identified as potential biomarkers of cystic disease in the rat model. Conclusion: The findings of this study demonstrate the potential of metabolomics to further investigate kidney disease. This paper reports on a method for non-targeted metabolite profiling of rat urine, using the approach to investigate the cystic phenotype of the Lewis polycystic kidney rat. The findings of this study demonstrate the potential of metabolomics to investigate kidney disease.