Abstract: Multicellular tumor spheroids (MCTS) can simulate the structure and metabolic characteristics of tumors in vivo, which is of great significance to study the metabolic phenotype of tumor cells and the mechanism of drug intervention. In this study, esophageal cancer MCTS were constructed, and MCTS frozen sections were prepared after treated with different formulations of paclitaxel (PTX) including common PTX injection, PTX liposome and albumin bound PTX. MCTS mass spectrometry imaging analysis method was established by using air flow assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI). The visualization of the permeation and enrichment process of PTX in MCTs after PTX treatment was realized, and the spatially resolved metabolomics of PTX injection group was studied. The results showed that the permeation and enrichment behavior of PTX in MCTs model were related to the formulations. The changes of endogenous metabolites in MCTs of esophageal cancer after treated with PTX injection had temporal and spatial characteristics. The metabolic changes of MCTS during the initial 0-4 hours were dominated by the down-regulation of middle-high polarity metabolites and some lipids in the central region of MCTS, while the metabolic changes of MCTS during 8-72 hours were mainly up-regulated by lipid metabolites in the peripheral region of MCTS. The combination of in vivo tumor-associated MCTs model with label free, highly sensitive and high coverage mass spectrometry imaging technology provided a new method and strategy for the study of pharmacometabolomics.