The lipid composition of cell membranes undergoes modifications during cellular differentiation (e.g. epithelial and blood cells) and also in diseased state (e.g. cancer progression). Therefore tissue- and/or tumour-specific properties (e.g. fluidity) and composition (e.g. phospholipid species) can be explored to improve selectivity by peptide-based therapeutics, as peptides can distinguish specific lipids, sterols, or prefer particular membrane properties. Our aim is to decipher the lipid composition of healthy and cancerous cell membranes to identify disease biomarkers and design peptides that can be used as diagnostic tools or as therapeutics. Using shotgun lipidomics approach, we have characterized the total lipid composition of a panel of tumour cells and healthy cells from a range of tissues. We also developed a method to determine the outer phospholipid layer of these cells by using a combination of enzymatic assays followed by mass spectrometry. By characterising the cell membrane of diverse cell types and investigating the activity of membrane active peptides, we hope to develop better peptide therapeutics in the future.