Background: In this era of precision medicine, the deep and comprehensive characterization of tumor phenotypes will lead to therapeutic strategies beyond classical factors such as primary sites or anatomical staging. Recently, “-omics” approached have enlightened our knowledge of tumor biology. Such approaches have been extensively implemented in order to provide biomarkers for monitoring of the disease as well as to improve readouts of therapeutic impact. The application of metabolomics to the study of cancer is especially beneficial, since it reflects the biochemical consequences of many cancer type-specific pathophysiological processes. Here, we characterize metabolic profiles of colon and ovarian cancer cell lines to provide broader insight into differentiating metabolic processes for prospective drug development and clinical screening. Methods: We applied non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography and gas chromatography for the metabolic phenotyping of four cancer cell lines: two from colon cancer (HCT15, HCT116) and two from ovarian cancer (OVCAR3, SKOV3). We used the MetaP server for statistical data analysis. Results: A total of 225 metabolites were detected in all four cell lines; 67 of these molecules significantly discriminated colon cancer from ovarian cancer cells. Metabolic signatures revealed in our study suggest elevated tricarboxylic acid cycle and lipid metabolism in ovarian cancer cell lines, as well as increased β-oxidation and urea cycle metabolism in colon cancer cell lines. Conclusions: Our study provides a panel of distinct metabolic fingerprints between colon and ovarian cancer cell lines. These may serve as potential drug targets, and now can be evaluated further in primary cells, biofluids, and tissue samples for biomarker purposes.