Inflammatory bowel disease (IBD) is highly prevalent globally and, in the majority of cases, remains asymptomatic during its initial stages. The gastrointestinal microbiota secretes volatile organic compounds (VOCs), and their composition alters in IBD. The examination of VOCs could prove beneficial in complementing diagnostic techniques to facilitate the early identification of IBD risk. In this protocol, a model of sodium dextran sulfate (DSS)-induced colitis in rats was successfully implemented for the non-invasive metabolomic assessment of different stages of inflammation. Headspace-gas chromatography-mass spectrometry (HS-GC-MS) was used as a non-invasive method for inflammation assessment at early and remission stages. The disease activity index (DAI) and histological method were employed to assess intestinal inflammation. The HS-GC-MS method demonstrated high sensitivity to intestine inflammation, confirmed by DAI and histology assay, in the acute and remission stages, identifying changes in the relative content of VOCs in stools. HS-GC-MS may be a useful and non-invasive method for IBD diagnostics and therapy effectiveness control. Key features • Experiments performed in vivo to better control DSS-induced colon damage and to aid the study of IBD development in humans. • Optimized for the following organisms: Wistar rats and C57BL/6 mice. • An easily assessed disease activity index (DAI) (weight loss, stool consistency, degree of fecal occult blood) and histopathological examination are suggested for additional IBD confirmation. • Enables VOC testing with relatively small stool samples.