Liposomal drug delivery systems are designed to avoid the body's natural clearance machinery and are typically achieved by incorporation lipid-anchored poly(ethylene glycol) (PEG) polymers. When preparing such liposomes it is intrinsically assumed that all available PEG-lipid is incorporated evenly into every liposome of a preparation, however this assumption remains unchallenged. Thus, a quantitative understanding of how PEG-lipid incorporation conditions affect the PEG density on individual liposomes can provide novel insights on how to optimize an important parameter of liposomal circulation kinetics and therapeutic efficacy. Here a sensitive fluorescence-based single liposome assay is employed that allows, for the first time, the quantification of the PEG-lipid density on individual liposomes. The assay is used to demonstrate that incubation time and temperature, liposome membrane lipid saturation state and PEG anchoring moiety all affect the effective PEG surface density. Furthermore, the unique ability of the assay to investigate single liposomes within the ensemble, allows to quantify significant variations in PEG surface density between individual liposomes. Thus, the assay provides crucial insights on how to improve both the overall PEG surface density and reduce the liposome-to-liposome variation, facilitating the design and development of more uniform, controllable, and efficacious PEG-lipid containing liposomal drug delivery systems.