Abstract: Cell metabolomics is now becoming an invaluable tool to gain mechanistic insight into biological processes. Carnitine and acylcarnintines play important roles in fatty acid oxidation. A method for metabolic profiling of acylcarnitines in glioma cells was developed herein by liquid chromatography tandem mass spectrometry (LC-MS/MS). The cell samples (5×106 ) were quenched by -80 ℃ 100% methanol and extracted with 80% methanol aqueous solution by freeze-thaw cycles. Separation and identification were performed using UPLC/Q-Exactive HRMS with electrospray ionization in positive mode. Firstly, the acylcarnitines reported in the HMDB database and literatures were selected from the full scan data and then chosen as precursor ions to be performed by parallel reaction monitoring (PRM) acquisition. The characteristic product ions of acylcarnitines at m/z 60.080 8, 85.028 4, 144.101 9, and the neutral loss of 59.073 5 Da were used to screen potential acylcarnitines. The metabolites identified as acylcarnitines should fulfill the following conditions: (1) The mass tolerance of precursor ion is within 5×10^-6. (2) More than three fragment features are detected at mass tolerance of 10×10^-6, and retention behavior of these fragment ions should match with the precursor ion. (3) The characteristic product ion of m/z 85.028 4 is prerequisite as it is the most stable fragment for acylcarnitines. As a result, a total of 17 acylcarnitines were identified in the U87MG malignant cells (GCs), U87MG stem-like cells (SLCs) and U87MG stem-like cell differentiation cells (SLCDCs), including seven short-, four medium and six long-chain acylcarnitines. Then 17 acylcarnitines was profiled using UPLC/Qtrap operated in the multiple reaction monitoring (MRM) mode, which enabled rapid quantification of 14 acylcarnitines within 15 min. The method afforded satisfactory results in terms of sensitivity, specificity, precision as well as accuracy. The results showed that the levels of carnitine and most acylcarnitines in SLCDCs and GCs were up-egulated compared with SLCs, which suggest that mitochondrial energy metabolism alterations associated with SLC self-renewal and differentiation. This study can promote the discovery of acylcarnitine-related metabolic reprogramming processes at the cellular level and may provide perspectives for anti-tumor research based on cellular energy metabolism.