Perfringolysin O (PFO) is a cholesterol dependent cytolysin (CDC) secreted by Clostridium perfringens, which forms pores in cholesterol containing membranes. CDCs are part of the larger Membrane attack complex-Perforin/CDC (MACPF/CDC) superfamily, containing pore formers responsible for controlling infectious disease and cancer in humans. CDCs are secreted as soluble hydrophilic monomers which oligomerise on lipid bilayers, ultimately forming bilayer spanning closed ring or open arc-shaped β-barrel pores. Perfringolysin O (PFO) was the first CDC to have its crystallographic structure resolved in its soluble monomeric form and has since become the prototypical CDC for investigating pore-forming mechanism. Previous studies on PFO have revealed a general outline of the steps involved in CDC pore formation; recognition of cholesterol and membrane binding, oligomerisation and ultimately membrane insertion to form large amphipathic pores. These steps have been elucidated using bulk assays and static imaging techniques such as electron microscopy or atomic force microscopy, however key mechanistic details remain uncharacterised due to the lack of time resolved data at a single pore level. Here we present a novel assay using microfluidics and total internal reflection microscopy to track PFO pore formation dynamics. Fluorescently labelled PFO and dye encapsulating liposomes were employed in conjunction to measure the kinetics of PFO binding from solution, nucleation, and oligomerisation on the surface of cholesterol containing liposomes. By visualising fluorescent dye release from our liposomes, we were able to determine the number of molecules necessary for an oligomer to insert and form a bilayer spanning pore. This information was collected from a large number of liposomes and used to build a mathematical model to quantify parameters controlling PFO accumulation and insertion kinetics on liposomes. We have also demonstrated how a different membrane composition affects these parameters by performing the assay on virus-like particles derived from HIV. This also displayed the adaptability of the assay which can be applied to different membranes and other pore forming proteins.