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[Display omitted] • Lipidating salmon calcitonin (sCal) alters its biophysical characteristics, including oligomer size, hydrophobicity and membrane activity. • Fluorescent imaging of single GUVs determines peptide mediated reporter dye influx behavior as either graded or All-or-None mechanism. • Modulating the number of hydrocarbons constituting the lipidation moieties determines the membrane permeation mechanism. • By increasing the lipid chain length lipidated of sCal goes from displaying smaller membrane perturbations to a peptide pore formation mechanism. • Effective membrane translocation of lipidated salmon calcitonin requires a peptide mediated pore forming mechanism. A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure–function relations could be pivotal for optimizing future peptide design strategies. [ABSTRACT FROM AUTHOR]