Exosomes are subcellular nanoparticles (50–200 nm in size) that are released from cells through a fusion of multicellular bodies with plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor exosomes have also been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and the need exists for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for isolation of large quantities of pure and sterile exosomes from cell culture media. Exosomes from cancer cell lines representing the most prevalent cancer types including PC3 (ATCC® CRL-1435™) and LNCaP (ATCC® CRL-1740™) prostate cells, HCT116 (ATCC® CCL-247™) colorectal cells, MDA-MB-231 (ATCC® HTB-26™) breast cells, A549 (ATCC® CCL-185™) lung cells, and U87-MG (ATCC® HTB-14™) glioblastoma cells were isolated and characterized in this study. We employed a stringent quality control approach in order to define and characterize these exosomes. Our method gave us high yield of >1×1010 exosomes/mL and average protein equivalent of 2 mg/mL. Our data demonstrated expression of a number of different exosome proteins including tetraspanin confirmed through Western blotting analysis. Isolated exosomes had a median size of around 102 nm through nanoparticle tracking analysis (NTA). Our western blot data showed differential protein marker expression of CD63, CD81, CD9, flotillin-1, and TSG101 protein and this expression profile is unique in differing tumor cell lines. We show the functionality of these exosomes through cellular uptake, anchorage-independent growth assay, and angiogenesis assay in this study. We also evaluated the quality and integrity of RNA from these purified exosomes and the RNA information demonstrates the presence of small RNAs (20-200nt) as expected; these RNA could be useful in biomarker studies. We show that exosomes isolated by our method not only had high exosome yield and quality but also were functional in nature and thereby make ideal reference standards/controls in the field of cancer research and diagnostics development.