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Modern diagnostics of acute myeloid leukemia (AML) lack rapid, label-free methods that provide information about the metabolic state of cells. Raman spectroscopy could fill this gap and enrich the diagnostic process with new data. Raman spectra serve as "molecular fingerprints" characterizing the biochemical composition of complex cellular structures. The study aimed to identify the spectroscopic Raman signatures of molecular changes induced by IDH1/2 oncogenes in AML models and to analyze the effect of a single mutation on the molecular structure of leukemia cells in vitro. Somatic mutations in the IDH1/2 gene are among the most common in adult AML patients. The effect on energy metabolism and the cell cycle make IDH mutants an ideal candidate for analysis with Raman spectroscopy. The HEL and HL-60 leukemic cell lines were retrovirally transduced with the IDH1//R132H, IDH2/R140Q, and IDH2/R172K constructs in pMIG vector. Cells were imaged using WITec Alpha 300 confocal Raman system. Recorded Raman images were subjected to clustering analysis using the k-means (KMCA) method, and then principal components analysis (PCA) and partial least squares discrimination (PLS-DA) were done on averaged spectra of cells and selected subcellular regions. PCA analysis showed that cells with mutated IDH1/2 genes showed differences in the content of proteins and nucleic acids compared to control cells. The most significant differences were observed in the Raman spectra of cells with the IDH1 R132H mutation. Using the PLS-DA method, a mathematical model was built to distinguish mutant transgenic cells from wild-type IDH1 and IDH2 cells. The obtained results confirm the effectiveness of Raman spectroscopy in identifying changes between mutant and wild-type forms of IDH1/2. Potential applications of Raman spectroscopy could be monitoring the progress of targeted therapy or action of specific inhibitors. To improve and optimize the method, more data ought to be collected for machine learning. This work was supported by „Label-free and rapid optical imaging, detection and sorting of leukemia cells" project carried out within the Team-Net program of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. [ABSTRACT FROM AUTHOR]