CD81 is a membrane protein of the tetraspanin family present in the tetraspanin-enriched microdomains (TEMs). CD81 is incorporated in the surface of both Human Immunodeficiency Virus type 1 (HIV-1) virions and extracellular vesicles (EVs). Using this feature a recombinant CD81 variant, incorporating a hexahistidine tag (His-tag) within its variable region, was designed to enable surface peptide display in HIV-1 Gag::eGFP virus-like particles (VLPs) and EVs. Immunofluorescence and immunogold analyses confirmed the correct localization of CD81-His-tag in the plasma membrane and its incorporation into both VLPs and EVs. However, co-expression of CD81-His-tag with Gag::eGFP led to a significant reduction (6.8-fold) in Gag::eGFP VLP production, that could be attributed to the overexpression burden associated to the co-expression of two recombinant proteins through transient transfection. Additionally, to study the compensation effect on the production and functionalization of VLPs and EVs, shRNA-mediated knockdowns were performed targeting proteins known to interact with CD81 in TEMs, including CD9, CD63, EWI-2, and EWI-F. The compensation effect on CD81-His-tag caused by shCD9 and shCD81-WT knockdowns allowed to maintain the levels of CD81-His-tag per particle while increasing particle production. These findings contribute to the development of engineered vesicles as platforms for peptide display in drug delivery, vaccines, and therapeutic applications.