Identifying metabolites in model organisms is critical for many areas of biology, including unravelling disease aetiology or elucidating functions of putative enzymes. Even now, hundreds of predicted metabolic genes inSaccharomyces cerevisiaeremain uncharacterized, indicating that our understanding of metabolism is far from complete even in well-characterized organisms. While untargeted high-resolution mass spectrometry (HRMS) enables the detection of thousands of features per analysis, many of these have a non-biological origin. Stable isotope labelling (SIL) approaches can serve as credentialing strategies to distinguish biologically relevant features from background signals, but implementing these experiments at large scale remains challenging. Here, we developed a SIL-based approach for high-throughput untargeted metabolomics inS. cerevisiae, including deep-48 well format-based cultivation and metabolite extraction, building on the peak annotation and verification engine (PAVE) tool. Aqueous and nonpolar extracts were analysed using HILIC and RP liquid chromatography, respectively, coupled to Orbitrap Q Exactive HF mass spectrometry. Of the approximately 37,000 total detected features, only 3–7% of the features were credentialed and used for data analysis with open-source software such as MS-DIAL, MetFrag, Shinyscreen, SIRIUS CSI:FingerID, and MetaboAnalyst, leading to the successful annotation of 198 metabolites using MS2database matching. Comparable metabolic profiles were observed for wild-type andsdh1Δ yeast strains grown in deep-48 well plates versus the classical shake flask format, including the expected increase in intracellular succinate concentration in thesdh1Δ strain. The described approach enables high-throughput yeast cultivation and credentialing-based untargeted metabolomics, providing a means to efficiently perform molecular phenotypic screens and help complete metabolic networks.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s00216-023-04724-5.