One of the major challenges in the drug development process is biodistribution across epithelia and intracellular drug targeting. Cellular membrane heterogeneity is one of the major drawbacks in developing efficient and sustainable drug delivery systems, which brings the need to study their interaction with lipids in order to unravel their mechanisms of action and improve their delivery capacities. Cell penetrating peptides (CPPs) are able to translocate almost any cell membrane carrying cargo molecules. However, different CPP use different entry mechanisms, which are often concentration-dependent and cargo-dependent. Being able to quantify the lipid affinity of CPP is of obvious importance and can be achieved by studying the partition extent of CPP into lipid bilayers. The partition constant (K(p) ) reflects the lipid-water partition extent. However, all currently available methodologies are only suitable to determine the partition of single molecules into lipid membranes or entities, being unsuitable to determine the partition of bimolecular or higher order supramolecular complexes. We derived and tested a mathematical model to determine the K(p) of supramolecular CPP-cargo complexes from fluorescence spectroscopy data, using DNA oligomers as a model cargo. As a proof-of-concept example, the partition extent of two new membrane active peptides derived from dengue virus capsid protein (DENV C protein) with potential CPP properties, in both scenarios (free peptide and complexed with a molecular cargo), were tested. We were able to identify the lipid affinity of these CPP:DNA complexes, thus gaining valuable insights into better CPP formulations. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.