Stable isotopes have frequently been used to study metabolic processes in live cells both in vitro and in vivo. Glutamine, the most abundant amino acid in human blood, plays multiple roles in cellular metabolism by contributing to the production of nucleotides, lipids, glutathione, and other amino acids. It also supports energy production via anaplerosis of tricarboxylic acid cycle intermediates. While ¹³C-glutamine has been extensively employed to study glutamine metabolism in various cell types, detailed analyses of specific lipids derived from ¹³C-glutamine via the reductive carboxylation pathway are limited. In this protocol, we present a detailed procedure to investigate glutamine metabolism in human glioblastoma (GBM) cells by conducting ¹³C-glutamine tracing coupled with untargeted metabolomics analysis using liquid chromatography–mass spectrometry (LC–MS/MS). The method includes step-by-step instructions for the extraction and detection of polar metabolites and long-chain fatty acids (LCFAs) derived from ¹³C-glutamine in GBM cells. Notably, this approach enables the distinction between isomers of two monounsaturated FAs with identical masses: palmitoleic acid (16:1n-7) (cis-9-hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-hexadecenoic acid) derived from ¹³C-glutamine through the reductive carboxylation process. In addition, using this protocol, we also unveil previously unknown metabolic alterations in GBM cells following lysosome inhibition by the antipsychotic drug pimozide.