It has been shown from the isolation and characterization of exosomes from cell culture media supplemented with fetal bovine serum that both their quality and purity are affected. The high abundance of serum proteins, including bovine cell derived exosomes, is also a potential source of contaminants, which may result in appreciable yields of impure exosomes, thereby leading to artifacts. Isolation and characterization of exosomes from cells maintained under serum-free conditions should therefore ensure the high quality necessary for medical applications. To meet this end, the present study aimed to characterize exosomes released from THP-1 macrophages cultured in serum-free, ultra-centrifuged medium upon infection with the human pathogen Mycobacterium tuberculosis (Mtb). Macrophages differentiated from the human cell line THP-1 were infected at a multiplicity of infection (MOI) of 5. Macrophages were cultivated in CellGenix ® GMP DC serum-free ultra-centrifuged medium for 4, 24 and 48 h at 37˚C in a humidified atmosphere with 5% CO 2 . Total exosome isolation reagent was used to extract the exosomes from the cell culture supernatants of naïve and Mtb-infected THP-1 macrophages. The size and purity of the exosomes isolated were subsequently assessed by various methods, including nanoparticle tracking analysis, flow cytometry, MACSPlex exosome analysis, and western blotting. The serum-free, ultra-centrifuged medium was found to support the proliferation of the THP-1 cells successfully. The nanoparticle tracking analysis data revealed that the majority of the isolated particles were within the size range of exosomes (i.e., 30-150 nM). The MACSPlex exosome analysis confirmed the expression of the exosomal markers, CD9, CD63 and CD81. Furthermore, western blot analysis of the isolated exosomes indicated the presence of CD9, CD63, CD81 and lysosomal associated membrane protein-1 (LAMP-1), and also confirmed the absence of Mtb proteins. Taken together, these data provide evidence that serum-free, ultra-centrifuged CellGenix ® GMP DC medium is suitable for application in exosome research, and may significantly advance such studies. Therefore, the use of serum-free medium for exosome isolation purposes could offer considerable advantages, and constitute a significant improvement in the growing field of extracellular vesicle research. The use of more sensitive methods represents an advance that will enable researchers to rule out the presence of Mtb pathogenic proteins in exosomes isolated from infected serum-free cell cultures.