Biomimetic nanosystems and vesicles have arisen as a novel approach to design vesicular transport systems with diverse therapeutic potential. The ‘biomimetic’ strategy involves the integration of cell membrane components into lipid bilayers, conferring them with biological properties originating from the cell of origin. Until now, most studies have primarily focused on the evaluation of the biological activity and function of different biomimetic nanosystems with limited exploration of the engineering parameters selected and little characterization of their features at the molecular level. This study aimed to address this knowledge gap by describing a preparation method for biomimetic lipid vesicles using traditional liposome fabrication principles and cellular components exclusively derived from glioblastoma (GL261) cell membrane proteins. Critical engineering parameters were studied, such as bilayer lipid and cholesterol content, the degree of surface PEGylation and some processing aspects like purification and quantification. Following fabrication, the GL261-derived vesicles underwent purification using size exclusion chromatography to separate unbound proteins from the vesicles. Subsequently, the GL261-derived vesicles were characterized by cryo-EM and differential scanning calorimetry (DSC) to assess their morphological and thermal properties, respectively. Both cholesterol and PEGylated lipid content played an important role on the structural and colloidal features of the biomimetic vesicles (BV). Mass spectroscopy (LC-MS/MS) revealed the proteomic signature of the fabricated vesicles at the molecular level. Collectively, these findings advance the rational engineering of BV, and offer an in-depth proteomic framework that reveals their molecular identity and functional potential. By connecting the design principles of fabrication with the molecular features of the vesicles, this study paves the way for next-generation biomimetic platforms for cancer chemotherapy, immunomodulation and cancer vaccination.