Daptomycin (DAP) is a cyclic lipopeptide antibiotic used for the treatment of certain Staphylococcus aureus infections. Although rare, strains have been isolated that are DAP resistant. These strains usually have mutations in mprF, a gene encoding a membrane protein with both lysylphosphatidylglycerol (LPG) synthase and flippase activities. Because ΔmprF strains have increased DAP susceptibility, the mechanism of resistance is not likely due to a loss of mprF function. In this study, we developed an LC-MS assay to examine the effect of different mprF mutations on the ratio of phosphatidylglycerol (PG) to LPG in the membrane. Our assay demonstrated that some, but not all, mutations in the flippase and synthase domains result in small but reproducible increases in the proportion of LPG relative to PG. Techniques described herein represent a higher throughput and more sensitive method for measuring relative phospholipids levels. These results offer guidance in the understanding of how mprF confers DAP resistance; namely, mprF-mediated resistance may be through more than one mechanism, including increased overall LPG synthesis and increased LPG present on the outer leaflet of the cytoplasmic membrane.