The term metabolomics refers to the systematic identification and quantification of all metabolites in certain organisms or biological samples. Currently, two complementary approaches are used in metabolic research: metabolic profiling and metabolic fingerprinting. Separation and high-resolution techniques for metabolite identification have been used in metabolomic analyses. After the raw spectra have been obtained using analytical techniques, preprocessing of the raw data is required before statistical analysis of the data. Statistical analyses can be univariate or multivariate. The aim of this study was to investigate the influence of compound (E)-3-amino-5-(3-(3- bromophenyl)acryloyl)-N-(3-chloro-2-methylphenyl)-6-methylthieno[2,3-b] pyridine-2-carboxamide (hereinafter thieno[2,3-b]pyridine) on the metabolism of two types of breast cancer cells MCF-7 and MDA- MB-231. Agilent MassHunter Qualitative Analysis software was used for peak definition, RT time alignment, and peak annotation and integration. MetaboAnalyst, a platform dedicated to metabolomic data analysis, was used to analyze differentially expressed metabolites across the two cell lines. The results of the analysis showed that the treatment of cells with thieno[2,3-b]pyridine had a great impact on glucose metabolism, that is, energy cycles in the cell, especially on glycolysis, gluconeogenesis, pyruvate metabolism, the occurrence of Warburg effect, and inositol metabolism. Thieno[2,3-b]pyridine showed greater cytotoxicity in MDA-MB-231 cells than in MCF-7 cells.