Assays on vesicle aqueous content leakage are widely used in the study of peptide-lipid interactions. We found this assay to be affected by the mode of mixing vesicle and peptide solutions. This effect can lead to artifactual conclusions regarding the lytic activity of peptides. We demonstrate that the source of this artifact is that fast (millisecond range) peptide-membrane association creates a nonhomogeneous distribution which exists sufficiently long after peptide addition to markedly alter the course of leakage. Mixing problems can be overcome by using a stopped flow apparatus. It can also be diminished by switching to injecting a small volume of vesicle suspension into a large volume of peptide solution. Mixing rates are rarely considered in literature reports of peptide effects on liposomes. The same artifacts can also take place in a number of other assays of the activity of membrane active peptides on liposomes and thus their consideration is of general importance.