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Detecting molecules involved in cancer is critical for cancer research and diagnostics. To achieve this goal, sensitive protein detection is essential to improving the chances of finding, verifying, validating and developing valuable biomarkers. Extracellular vesicles (EVs) are membrane-enclosed nanometer-size structures that can transport macromolecular information between cells. While they play an essential role in cell-to-cell communication, they may also prove important as biomarkers for minimally invasive detection of cancer. In this doctoral thesis the aim was to establish protocols for proteome analysis of EVs, specifically to identify combinations of surface proteins on the EVs by labeling surface proteins followed by protein identification via mass spectrometry. Also, using the proximity ligation and extension assays the challenges have been met of discovering and validating proteomics biomarkers with very low amounts of EVs. In paper I the aim was to develop a detection method and protocol combining high-resolution mass spectrometry with solid-phase- and Exo PLAs for identifying surface proteins on EVs with relevance in prostate cancer. The protocol allowed identification of more than 1,000 surface proteins, many not previously reported to be carried by EVs. In Paper II we used five protein assay panels consisting of more than 400 proteins to assess and analyze the proteomics profiles of EVs isolated from four different gastric cancer cell lines. The data identified 39 proteins with medium or high expression levels in EVs from gastric cancer cell lines, which were not expressed or are only present at low concentrations in control EVs from seminal fluid. In Paper III we analyzed and measured thymidine kinase 1 enzyme activity in EVs purified from seminal fluids from healthy individuals and from normal and prostate cancer cell lines. Thymidine kinase 1 is a cell cycle-dependent enzyme and a biomarker for cell proliferation. The results indicate a correlation of TK1 enzyme activates with the aggressiveness of the tumor cell lines and higher enzyme activity was recorded for EVs isolated from p53 null and mutated cell lines compared to cells with wild-type p53. Paper IV describes a high-throughput approach using in situ proximity ligation assays (in situ PLA) to investigate protein interactions and post-translational modifications in the HaCAT cell line. In situ PLA was combined with automated microscopy and computerized analysis to evaluate phosphorylation and protein interaction along with subcellular features in response to drug treatment. In summary, the focus of this Ph.D. thesis has been to adopt a variety of proteomic techniques for investigating EVs as biomarkers in health and disease.