Faecal metabolite profiling, though in its infancy, allows for investigation of complex metabolic interactions between gastrointestinal infections or diseases and host health. In the present study, we describe a faecal metabolite extraction method for untargeted gas chromatography-mass spectrometry (GC-MS) analysis using Cryptosporidium positive and negative human faecal samples. The extraction method takes into account the varying faecal consistencies and quantities received for clinical diagnosis. Optimisation was carried out using different extraction solvents and on three different faecal quantities to determine the minimum amount of faecal sample required. The method was validated by untargeted GC-MS analysis on 8 Cryptosporidium positive and 8 Cryptosporidium negative human faecal samples, extracted using the optimised conditions. The method showed good extraction reproducibility with a relative standard deviation of 9.14%. Multivariate analysis of the GC-MS generated dataset showed distinct differences between profiles of Cryptosporidium positive and Cryptosporidium negative samples. The most notable differences included changes in amino acid, nitrogen and energy metabolism, demonstrating the association of infection with Cryptosporidium and altered permeability of the small intestine.