The actual utility of capillary electrophoresis-mass spectrometry (CE-MS) for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic profiling study for biomarker discovery by CE-MS was performed, using pooled human plasma samples with spiked biomarkers. Two studies have been carried out in this work. Focus of study I was on comparing two sets of plasma samples, in which one set (class I) was spiked with five isotope-labeled compounds, whereas another set (class II) was spiked with six different isotope-labeled compounds. In study II, focus was also on comparing two sets of plasma samples, however, the isotope-labeled compounds were spiked to both class I and class II samples but with concentrations which differ by a factor two between both classes (with one compound absent in each class). The aim was to determine whether CEMS-based metabolomics could reveal the spiked biomarkers as the main classifiers, applying two different data analysis software tools (MetaboAnalyst and Matlab). Unsupervised analysis of the recorded metabolic profiles revealed a clear distinction between class I and class II plasma samples in both studies. This classification was mainly attributed to the spiked isotope-labeled compounds, thereby emphasizing the utility of CE-MS for biomarker discovery.