We have engineered and validated a synthetic peptide (Vn96*), which has specific affinity for canonical heat shock proteins (HSPs), as a tool for the rapid isolation of exosomes or extracellular microvesicles (together referred to herein as eMVs) from cell culture media, human and animal body fluids. Most of the available methods of eMV isolation are based on physical characteristics (density and/or size separations) or multiple antibody affinity-based purification, which are not compatible with platform versatile high-throughput down-stream clinical applications. Given the presence of increased surface expression of HSPs on eMVs from pathological cells (e.g. cancers, acute ischemic stroke, allergy, diabetes, hypertension, infection, coronary artery disease), the discovery and validation of the Vn96 peptide as a tool to enrich “pathological” eMVs may have the potential for immediate and future diagnostic applications. Furthermore, the peptide chemistry for adaptation to various clinical assay platforms is well understood, robust and cost-effective. Methods: The eMV fractions isolated from conditioned media, plasma and urine using Vn96 peptide were compared to those obtained by ultracentrifugation or using a commercial purification reagent. These validation analyses were performed using nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, immunoblotting, next-generation sequencing of miRNA cargo and proteome-based cellular component ontology analysis. Results: Our preliminary clinical data resulting from analyses of post-digital rectal examination urinary Vn96-captured eMVs from prostate cancer patients and healthy controls (with informed consent and medical ethics committee approval) demonstrated that we can detect and measure (via qPCR) a number of miRNAs previously identified by others in plasma and urine sediments where their expression was shown to be up-regulated in prostate cancer patients compared to healthy individuals. Similarly, mRNA analyses of Vn96-captured urinary eMVs demonstrated the presence of several known prostate cancer-associated markers (e.g. PSA, PCA-3, FOLH1, PSMA, SPINK1, PSCA, CD24, GOLM1, ANXA3, TMPRSS2-ERG fusion, SLC45A3). PCA-3/PSA mRNA ratios measured using Vn96 eMV RNA yielded improved specificity and accuracy for detecting prostate cancer compared to ratios measured using standard urine sedimentary procedures. Summary/conclusion: Together, our results suggest that Vn96-mediated capture of eMVs can improve disease diagnosis in the clinical setting.