Abstract Introduction. Tissue Microarrays (TMAs), widely utilized in the field of pathology, have now found a powerful ally in Image-based Spatial Transcriptomics (ST). By analyzing various gene expression data with high resolution, image-based ST data on TMA can provide the heterogeneous patterns of tumor microenvironment in multiple samples. However, an efficient method for processing multiple samples post-data acquisition is still under development. A streamlined process would expedite the discovery of spatial gene expression patterns, thereby enhancing our understanding of the tumor microenvironment and its implications for cancer diagnostics and treatment strategies. Methods. Our automated pipeline is initiated by automatic segmentation of every core in the whole TMA, derived from the processed output of the MERSCOPE or Xenium platform. Then, it subsequently removes QC failed cells unrelated to any core. Each core receives sequential naming and undergoes automated cell type mapping utilizing a reference single-cell RNAseq data. Additionally, the pipeline computes neighborhood enrichment between cell types, providing a nuanced comprehension of spatial relationships and interactions among diverse cell populations within the tissue microenvironment from multiple samples on the TMA. Results. The automated pipeline we've developed provides several key advantages. It enables the simultaneous analysis of multiple tissue cores, effectively minimizing batch effects and ensuring the reliability of results across diverse tissue samples. Furthermore, by automating core separation, labeling, and cell type mapping, our pipeline significantly streamlines the time and effort required for TMA data analysis. This cost-effective approach allows researchers to optimize resource allocation, making our automated pipeline a valuable tool in cancer research. It facilitates the exploration of gene expression patterns within specific tissue regions, ultimately contributing to the advancement of our understanding of cancer biology. Conclusion. By seamlessly integrating TMA data with image-based ST technologies such as Xenium and MERSCOPE, we can perform comprehensive spatial transcriptomics analyses and provide detailed statistical information on cell type proportions within the tumor microenvironment. This automated pipeline not only ensures robust and reliable results across diverse tissue samples but also optimizes resource allocation by minimizing batch effects and reducing analysis time. This cost-effective solution empowers researchers to delve deeper into spatial gene expression patterns, enhancing our comprehension of the tumor microenvironment. Citation Format: Dongjoo Lee, Seungho Cook, Yeonjae Jung, Myunghyun Lim, Jae Eun Lee, Hyung-Jun Im, Daeseung Lee, Hongyoon Choi. Automated tumor microenvironment analysis for multiple samples by image-based spatial transcriptomics on tissue microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4948.