Abstract: Three-dimensional (3D) cancer cell culture model is a promising platform that is able to bridge the gap between animal models and two-dimensional (2D) cancer cell culture models. Compared to traditional two-dimensional cancer cells, three-dimensional tumor spheroids are capable of mimicking some characteristics of solid tumors in terms of spatial architecture and drug resistance mechanism. Mass spectrometry imaging (MSI) is a label-free powerful tool that can simultaneously provide mass spectra and images for hundreds of compounds. Increasing studies have used matrix-assisted laser desorption/ionization MSI to investigate the distributions of endogenous and exogenous compounds in multicellular tumor spheroids (MCTS). However, all these studies only focused on using vacuum MALDI to perform the experiments. To the best of our knowledge, no study has been reported on using atmospheric pressure MALDI to analyze the spatial distribution of endogenous metabolites in MCTS. Here, an analytical method based on an atmospheric pressure matrix-assisted laser desorption/ionization-quadrupole-exactive-focus mass spectrometry was established for detection of the spatial distribution of endogenous compounds in HCT116 colon carcinoma cell spheroids. Lipids were extracted and analyzed by using a HPLC system coupled with an orbitrap fusion tribrid mass spectrometer with a C18 column. Mass spectra were acquired at negative ionization mode by using three matrices including 9-aminoacridine, 2,5-dihydroxybenzoic acid and trans-2-3-(4-tert-butylphenyl)-2-methyl-2-propenylidenemalononitrile. The endogenous compounds were assigned by comparing the obtained MS information in three databases (metlin, lipidsearch and lipidmap) with the deviation of mass-to-charge ratio less than 1×10^-5. A total of 22 metabolites were analyzed including four metabolites belonging to tricarboxylic acid cycle and 18 lipids acting roles in biosynthesis and degradation of glycerophospholipids. The MSI results showed that these four metabolites in tricarboxylic acid cycle, including α-ketoglutarate, malate, oxaloacetate and succinate distributed in entire cell spheroids, while the distribution of 18 lipids showed spatial diversity. For instance, PGP(O-30∶3) and PGP(30∶2) located in centre region; PG(O-36∶1) distributed in outer region; and PE(18∶1/22∶6) located in entire zone. These results were further confirmed by the segmentation analysis revealing that mass spectra of the centre zone and the outer zone in cell spheroids were quite different. The study may improve the understanding of tumor microenvironment and offer molecular insights into cancer metabolism.