Hydrophobicity is an intrinsic property that relates to a chemical's tendency to partition between a polar and a nonpolar phase. Traditionally, hydrophobicity has been described by the logarithm of the octanol-water partition coefficient (log K-OW or log P). Immobilized artificial membrane high-performance liquid chromatography (IAM-HPLC) is an alternative method to determine hydrophobicity that may be more biologically relevant. This paper examines the robustness of an IAM-HPLC assay optimized using the IAM.PC.DD2 column to determine the retention factor (log k(IAM) ((pH 7.4))). The method has been shown to be robust following robustness testing of five compounds (log K-OW ranging from 0.29 to 6.03) assessed across five columns (which included three batches of stationary phase) and two HPLC systems.