Stable isotope-resolved metabolomics (SIRM) can provide metabolic conversion information of specific targets; it is a powerful tool for cell metabolism studies. The common analytical platform for SIRM is chromatography-mass spectrometry, which requires a large number of cells and is not suitable for precious rare cell analysis. To study a small number of cells, we established a novel SIRM method using chip-based nanoelectrospray mass spectrometry (MS). 13C-glutamine was taken as an example; the unlabeled and 13C-labeled cells were cultured and extracted in a 96-well plate and then directly injected into MS and analyzed in full scan mode and parallel reaction monitoring (PRM) mode targeting 44 glutamine-derived metabolites and their isotopologues. To define focused metabolite-related MS2 fragments produced in the PRM, a new strategy was proposed including MS2 exact m/z matching, MS2 false positive filtering, and MS2 fragment grouping to remove the interfering MS2 ions. In total, 292 and 349 pairs of paired MS2 ions were obtained in positive and negative ionization modes, respectively. By searching spectra databases, 31 targeted metabolites with their isotopologues were identified and their characteristic product ions were confirmed for MS2 quantification. The relative quantification was achieved by MS2 quantification, which showed better sensitivity and accuracy than common MS1-based quantification. Finally, this method was applied to isocitrate dehydrogenase I-mutated glioma cells for revealing the effects of triptolide on glioma cell metabolism using U-13C-glutamine as a labeling substrate.