Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered “impurities” in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.