Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.