Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles and provide a universal molecular cell-cell communication system. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support growth and invasion of tissues by tumor cells. EVs were purified from cell culture medium by ultracentrifugation followed by size exclusion chromatography with Exo-spin™ columns (Cell Guidance Systems Ltd). We performed proteomic analysis of five hypoxia/normoxia pairs of EV samples; each of these replicates for either hypoxia or normoxia was analyzed in duplicates. Obtained data were compared with proteomics analysis of corresponding cell culture media supernatants (SN) after EV removal with 100 000 g ultracentrifugation. We found that exposure of tumor cells to hypoxia (1% oxygen) induced EV secretion, altered EV sizes, and led to notable changes in the EV protein cargo in comparison to normoxia.