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Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann–Pick Type C (NPC) and Alzheimer's Disease1–4. Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol–rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan–deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP–binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.Giant plasma membrane vesicles serve as a means to monitor cell surface cholesterol.Increase in cholesterol efflux enhances giant plasma membrane vesicle formation.Microtubule stabilization and acetylation promote cholesterol efflux in NPC disease.Actin dynamics and Arl4c modulate lipid efflux and giant plasma membrane vesicles.