Anionic lipids, such as phosphatidylglycerol and phosphatidylserine, play important structural and functional roles in cell membranes. In particular, they appear to provide strong interactions with positively charged protein side chains to promote membrane localization of lipid binding domains and antimicrobial peptides, as well as to modulate the function of many membrane proteins. All-atom molecular dynamics simulations were used to explore the strength of these interactions and the impact they have on the ability of charged protein residues to penetrate into membranes. Using an analog of arginine and bilayers of pure phosphatidylcholine or mixtures with phosphatidylglycerol, we have computed the thermodynamics of charged side chain translocation, as well as the binding affinity of each lipid within the membrane. We found that arginine deforms the bilayer in a similar fashion, regardless of composition, and that the free energy profile for translocation is relatively unaffected by anionic lipids: the “neutralization” of the protein side chain does not reduce the large ∼20 kcal/mol barrier significantly. We decomposed these free energies to explain why anionic lipids do not play a significant role, with implications for the actions of many charged peptides and ion permeability. We also find that arginine binding to phosphatidylglycerol is more favorable by only ∼1 kcal/mol, suggesting that lipid binding domains and antimicrobial peptides likely require many charged side chains acting together to promote membrane localization.