Peptides are popular ligands to modulate intracellular therapeutic targets due to their small molecule like biophysical properties combined with high binding affinity and specificity. However, peptide delivery into their intracellular targets is still a limiting bottleneck. Many active peptides cannot enter cells, and the ones that enter cells often use endocytic pathways and get trapped inside the endosomal vesicles without reaching their cytosolic targets. Few peptides escape endosomes, but the exact mechanism of their endosomal release is not clearly understood. Therefore, we aim to investigate the molecular details involved in endosomal release by studying cell penetrating peptides (CPPs) with the ability to escape endosomes, and their membrane interactions using fluorescence spectroscopy methodologies and endosomal membrane models. In addition, lipidomic characterisation of the of early and late endosomes will be done for the first time to unravel the endosomal membrane properties facilitating the escape of CPPs. With this workflow we will decode the mechanisms of endosomal escape of CPPs via understanding both the peptide and membrane properties facilitating the escape; this knowledge will be applied in the design of peptide therapeutics with the ability to enter cells, escape endosomes, and reach their cytosolic targets. The poster demonstrates the conceptualisation of the assays and the investigation workflow of this study.