The endosomal sorting complexes required for transport (ESCRT) machinery drives membrane scission for diverse cellular functions that require budding away from the cytosol, including cell division and transmembrane receptor trafficking and degradation. The ESCRT machinery is also hijacked by retroviruses, such as HIV-1, to release virions from infected cells. The crucial roles of the ESCRTs in cellular physiology and viral disease make it imperative to understand the membrane scission mechanism. Current methodological limitations, namely, artifacts caused by overexpression of ESCRT subunits, obstruct our understanding of the spatiotemporal organization of the endogenous human ESCRT machinery. Here, we used CRISPR/Cas9-mediated knock-in to tag the critical ESCRT-I component tumor susceptibility 101 (Tsg101) with GFP at its native locus in two widely used human cell types, HeLa epithelial cells and Jurkat T cells. We validated this approach by assessing the function of these knock-in cell lines in cytokinesis, receptor degradation, and virus budding. Using this probe, we measured the incorporation of endogenous Tsg101 in released HIV-1 particles, supporting the notion that the ESCRT machinery initiates virus abscission by scaffolding early-acting ESCRT-I within the head of the budding virus. We anticipate that these validated cell lines will be a valuable tool for interrogating dynamics of the native human ESCRT machinery.