Ibuprofen (IBU) interacts with phosphatidylcholine membranes in three distinct steps as a function of concentration. In a first step (<10 μM), IBU electrostatically adsorbs to the lipid headgroups and gradually decreases the interfacial potential. This first step helps to facilitate the second step (10-300 μM), in which hydrophobic insertion of the drug occurs. The second step disrupts the packing of the lipid acyl chains and expands the area per lipid. In a final step, above 300 μM IBU, the lipid membrane begins to solubilize, resulting in a detergent-like effect. The results described herein were obtained by a combination of fluorescence binding assays, vibrational sum frequency spectroscopy, and Langmuir monolayer compression experiments. By introducing trimethylammonium-propane, phosphatidylglycerol, and phosphatidylethanolamine lipids as well as cholesterol, we demonstrated that both the chemistry of the lipid headgroups and the packing of lipid acyl chains can substantially influence the interactions between IBU and the membranes. Moreover, different membrane chemistries can alter particular steps in the binding interaction.