<p>Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) is a powerful technique suited for resolving complex mixtures of volatile organic compounds (VOCs). In this study, GC×GC-TOFMS was combined with an image-based chromatographic fingerprinting strategy to characterize the fecal volatilome of subjects affected by non-celiac gluten/wheat sensitivity (NCGWS) under dietary intervention. Fifty participants underwent a dietary protocol consisting of a gluten-free diet followed by gluten reintroduction while receiving probiotics or placebo. Headspace solid-phase microextraction (HS-SPME) conditions were specifically optimized to maximize the extraction coverage of volatiles, while a combined untargeted–targeted fingerprinting approach (<i>UT fingerprinting</i>) enabled the mapping of approximately 1000 volatile features in fecal samples. Chemometric analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), highlighted informative differences in VOC profiles induced by gluten reintroduction and probiotic administration. Around 270 VOCs were annotated, expanding the existing knowledge on the human fecal volatilome by 20%, predominantly comprising microbial fermentation-derived metabolites such as short-chain fatty acid esters, aldehydes, alcohols, terpenes, and aromatic hydrocarbons. PLS-DA models discriminated probiotic-treated individuals from placebo recipients with 89–90% accuracy, confirming the impact of probiotic supplementation on gut metabolic signatures. The integration of GC×GC-TOFMS and image-based chromatographic fingerprinting effectively captured subtle metabolic variations, demonstrating its suitability in biomarker discovery for dietary interventions and personalized nutrition studies.</p> Graphical abstract <p></p>