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This work aims to explore whether the laser tweezers Raman spectroscopy (LTRS) could be practical in separating adriamycin-resistant chronic myeloid leukemia cells K562\/adriamycin from its parental cells K562, and to explore the potential mechanisms. Detection of LTRS initially reflected the spectral differences caused by chemoresistance including bands assigned to carbohydrates, amino acid, protein, lipids, and nucleic acid. In addition, principal components analysis as well as the classification and regression trees algorithms showed that the specificity and sensitivity were above 90%. Moreover, the band data-based classification and regression tree model and receiver operating characteristic curve further determined some important bands and band intensity ratios to be reliable indexes in discriminating K562 chemoresistance status. Finally, we highlighted three metabolism pathways correlated with chemoresistance. 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