In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.