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Background Multiplex immunofluorescence (mIF) has emerged as a powerful tool in spatial biology that allows for profiling of protein expression within the tumor microenvironment including cell phenotyping the immune response. By enabling simultaneous detection of multiple biomarkers on a single formalin-fixed paraffin-embedded (FFPE) tissue section, mIF overcomes the limitations of traditional single-plex immunohistochemistry (IHC) and provides novel insights into cellular phenotypes, interactions, and spatial organization. This approach allows us to better understand tumor-immune dynamics and may help predict response to immunotherapy.Methods We first developed single plex IHC assays for each individual marker and then assembled them into multiplex panels. Fluorophore assignment and marker positioning were tested for optimal signal performance, followed by range and linearity testing to identify the optimal primary antibody concentration. Once the panels were finalized, we conducted analytical sensitivity screens across specific tumor indications, to evaluate and quantitate fluorophore emission and spatial complexity. Finally, analytical precision testing was performed to demonstrate the repeatability of the assay. These mIF panels are RUO validated and can be used to test retrospective clinical patient samples.Results In this study, we employed a tyramide signal amplification-based mIF workflow to evaluate several panels containing up to eight biomarkers, including T-cell markers (e.g. CD3, CD8, and FoxP3), immune checkpoint markers (e.g. PD-L1), and macrophage markers (e.g. CD68 and CD163), in both tumor and normal tissue samples. Multispectral imaging and machine learning-based image analysis enabled accurate cell segmentation, phenotyping, and spatial context analysis at subcellular resolution.Conclusions These findings underscore the value of mIF as an important tool for in-depth spatial characterization of the tumor immune microenvironment. The validated RUO panels provide a robust platform for biomarker discovery and translational research, with potential applications in companion diagnostics. Ongoing efforts aim to assess assay performance in clinical trial settings and explore its utility in predicting immunotherapy response.Ethics Approval Discovery procures Biospecimens from its source sites pursuant to protocols approved by an Institutional Review Board (IRB)/Ethics Committee (EC) in accordance with applicable country-specific regulatory requirements. Consent Statuses: Biospecimens may be categorized as one of the following consent statuses: Informed Consent: The subject has given written consent for participation. The consent meets all elements of informed consent as outlined in the Food and Drug Administration’s (FDA) Code of Federal Regulations (CFR)21 CFR 50.25, or the applicable local regulatory standard if outside the United States. Waiver of Consent: The IRB/EC has approved the use of Biospecimens and/or data without written subject consent. In order to receive IRB/EC approval for waiver of consent, the study must satisfy all elements of 45CFR 46.116(f), or the applicable local regulatory standard if outside the United States.