Enriched tumor epithelium, tumor-associated stroma, and whole tissue were collected by laser microdissection from thin sections across spatially separated levels of ten high-grade serous ovarian carcinomas (HGSOC) and analyzed by mass spectrometry. Unsupervised analyses of protein abundance data revealed independent clustering of enriched stroma and enriched tumor epithelium, with whole tumor tissue clustering driven by overall tumor “purity”. Comparing these data to previously defined prognostic HGSOC molecular subtypes revealed protein expression from tumor epithelium correlated with the differentiated subtype, whereas stromal proteins correlated with the mesenchymal subtype. Protein abundance in tumor epithelium and stroma exhibited decreased correlation in samples collected just hundreds of microns apart. These data reveal substantial tumor microenvironment protein heterogeneity that directly bears on prognostic signatures, biomarker discovery, and cancer pathophysiology, and underscore the need to enrich cellular subpopulations for expression profiling.