Fluorescence correlation spectroscopy (FCS) is a powerful method to investigate molecular interactions based on the variation of diffusion properties at the single-molecule level. This technique allows studying quantitatively the interaction of fluorescently labeled proteins/peptides with lipid vesicles. Here, we describe how to acquire and analyze FCS partition data in order to accurately determine the protein/peptide partition coefficients between the aqueous and lipid phases. It is shown that the recovery of unbiased partition coefficients from FCS partition curves (fractional amplitude of the bound species versus lipid concentration) requires considering explicitly the Poissonian loading of the lipid vesicles with the fluorescently labeled protein in order to account for the variable liposome brightness in each sample. Additionally, the impact of a trace amount of a fluorescent non-binding component on the partition curves determined by FCS is also discussed.