Milk phospholipids (PLs) are valuable dairy components and appear to impart human health benefits, including improved cognitive function in infants and adults. Furthermore, PLs have been shown to lower LDL cholesterol and improve intestinal barrier function. The commercial food industry uses other dietary sources of PLs, such as soy lecithin. However, it remains unclear whether dissimilar composition of PLs from different dietary sources convey the same benefits. The hypothesis of this work was that milk and soy PLs (MPLs and SPLs, respectively) will produce different physiological responses in cell culture. Our objective was to develop a cell-based method to compare the potential health benefits of milk and soy PLs to ultimately focus on inflammation. To improve stability of PLs in cell culture media, liposome structures made from milk and soy PLs (MPL and SPL, respectively) were prepared, optimized, and fully characterized. Large and stable unilamellar vesicles (LUVs) were attained with particle sizes of 232.1 ± 7.5 and 221.8 ± 10.0 nm in diameter and zeta potentials of −16.41 ± 2.49 and −28.01 ± 2.81 mV for MPL- and SPL-LUVs, respectively. Subsequently, 3T3-L1 adipocytes were treated with 0.05, 0.25, 0.5, and 1.5 mg/mL MPL- or SPL-LUVs and analyzed for changes in cell viability and cytotoxicity. The optimized conditions showed that the non-toxic, physiological range for cell culture was between 0.05 and 0.5 mg/mL. There also was a significant difference (P < 0.05) between cell viability of MPL- and SPL-LUVs treated cells at 0.25 and 0.5 mg/mL, suggesting that MPL-LUVs may have greater bioavailability compared with SPL-LUVs. These findings lead to our prediction that MPL-LUVs will have a greater anti-inflammatory effect than SPL-LUVs because of differences in PL composition, specifically sphingomyelin. To test this, adipocytes will be stimulated with bacterial endotoxins and characterized for NF-kB-mediated inflammation after treatment with LUVs. We propose that PL compositional differences may be the basis for differences in physiological response.