The pharmacological interaction between plasma proteins and pharmaceuticals is a relationship that will impact the systemic effects the drug will have in-vivo. In order to determine how much of the drug remains unbound to the plasma proteins equilibrium dialysis is commonly used. The accuracy of the equilibrium dialysis protein binding results is critical for some programs that rely on this data to move ahead with future studies or abandon the new drug or treatment entirely. Here, we report on the protein binding effects of plasma/serum age and the plasma anti-coagulant when propranolol is spiked in plasma/serum and the percent protein binding is determined. A prototype phospholipid removal plate was used for the extraction and MFLC-MS/MS was used for the analysis.