Abstract Glioblastoma (GBM) is an aggressive and almost universally fatal brain cancer. Glioblastoma tumor masses are highly vascularized and infiltrated with cells mediating both innate and adaptive immunity. The presence and organization of various immune cells within the tumor microenvironment varies between patients and within individual tumors. This variability in the immune landscape influences responses to different treatment modalities, including immunotherapies. Understanding reciprocal immune-tumor cell interactions will enhance design and implementation of novel therapies. Currently, review of GBM patient tissues by neuropathologists is done visually using single or low-plex biomarker panels whose complexity is constrained by limitations of traditional immunohistochemistry, including the lack of well characterized primary antibody clones, limited antibody selection due to species-dependent workflows, and inaccuracies such as false signal intensities introduced by autofluorescence (AF) and spectral overlap from traditional immunofluorescence imaging methods. Multiplex immunofluorescence (mIF) and multispectral imaging (MSI) allows for fast, accurate imaging on a single tissue section of up to eight fluorophores (using Phenoptics technology), cell type-specific AF spectra, and DAPI/Hoechst. It has been shown in other cancer types (i.e. lung cancer, breast cancer, melanoma) that spatial biology techniques including multiplex immunofluorescence is necessary to determine cellular densities and interactions in tissue context to better predict outcomes and stratify patient populations. Here, we have used pre-validated mIF kits that use Opal fluorophores together with the Vectra Polaris Automated Quantitative MSI system on human formalin-fixed, paraffin embedded GBM tissue samples to further characterize immune-tumor microenvironments, with consideration for spatial distributions of interacting cells. Pre-optimized immunofluorescent kits targeting immune cell subsets, checkpoint inhibitors, and other biomarkers have been shown to provide clinically relevant information in other cancer types (such as lung cancer and melanoma). Our study indicates that widespread use of these reagents, coupled with spatial analysis and complex phenotyping, would better characterize the complex immune landscape of tumors and enhance design and implementation of therapeutics for GBM. Citation Format: Agnes E. Haggerty, Najiba Mammadova, Michael E. Barish, Christine E. Brown. The importance of spatial analysis and multispectral imaging when evaluating tumor-immune interactions in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3876.