Previous studies have shown that metabolomics can be a useful tool to better understand the mechanisms of carcinogenesis; however, alterations in biochemical pathways that lead to bladder cancer (BC) development have hitherto not been fully investigated. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to unveil the metabolic alterations between low-grade and high-grade BC cultured cell lines. Multivariable analysis revealed a panel of metabolites responsible for the separation between the two tumorigenic cell lines. Significantly lower levels of fatty acids, including myristic, palmitic, and palmitoleic acids, were found in high-grade versus low-grade BC cells. Furthermore, significantly altered levels of some amino acids were observed between low- and high-grade BC, namely glycine, leucine, methionine, valine, and aspartic acid. This study successfully demonstrated the potential of metabolomic analysis to discriminate BC cells according to tumor aggressiveness. Moreover, these findings suggest that bladder tumorigenic cell lines of different grades disclose distinct metabolic profiles, mainly affecting fatty acid biosynthesis and amino acid metabolism to compensate for higher energetic needs.