Eruca sativa Mill. (rocket; Fam. Brassicaceae) is widely appreciated for its peculiar flavour and beneficial effects on human health. Glucosinolates (GSLs) and their enzymatic hydrolysis products, isothiocyanates (ITCs), are considered to be responsible for health-promoting effects and for sensory relevance in rocket, respectively. This study aimed at evaluating and comparing the metabolite profiles of rocket leaves collected at different phenological stages, to investigate the content evolution during cultivation. To minimise metabolic variability induced by environmental factors, plants were cultivated in an innovative growing system equipped with precision lighting and ventilation. A multi-platform metabolomics approach combining liquid chromatography–high-resolution mass spectrometry (LC–HRMS) and headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC–MS) was carried out for comprehensive coverage of non-volatile and volatile organic compounds (VOCs). To integrate data from both platforms, a multivariate data fusion strategy was used. Higher GSLs content was detected in the microgreens stage. In particular, glucoraphanin, glucoiberverin, glucoerucin, DMB-GLS, and 1,4-dimethoxyglucobrassicin were identified as biological markers of rocket microgreens. ITCs levels were found to increase in mature leaves. These findings suggest a dynamic modulation of secondary metabolism during the plant life cycle, possibly in response to different adaptation needs to environmental conditions. Our findings confirm the potential of microgreens as a functional food in promoting health and preventing chronic diseases and can also tailor rocket cultivation to maximise the production of beneficial metabolites and to improve selected sensorial features. © 2025 by the authors.

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