Based on ultra performance liquid chromatography coupled with ion trap-time of flight mass spectrometry(UPLC-IT-TOF MS),a non targeted metabolomics method was developed for the investigation on metabolite difference between human chronic myeloid leukemia cells and their adriamycin resistant cells,ie.K562 and K562-ADM.Specifically,impacts of disruption methods,extraction solvent systems and volumes on the extraction effects for metabolites in QC samples were examined.Results showed that the ultrasonic disruption method in an 800 μL of 80% methanol solvent system could obtain more feature peaks from leukemia cells with good repeatability and stability.Then,the established method was applied to investigate the metabolite difference between K562 and K562-ADM.The data was processed by principal component analysis(PCA) and orthogonal partial least squares discrimination analysis(OPLS-DA),and the significant differences between the metabolome of K562 and that of K562-ADM cells were obtained,in which forty metabolites significantly contributed to the differences,and they were mainly involved in amino acid metabolism, sphingolipid metabolism,purine metabolism and pyrimidine metabolism.The established method could be used to investigate the metabolite differences between K562 and K562-ADM,and further provide some help for future metabolomics researches on drug resistance of leukemia cells.