Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally employed technique that separates proteins on the basis of molecular weight (MW). However, membrane proteins are known to size anomalously on SDS-PAGE calibrated with conventional standards, an issue that complicates interpretation of protein identity, purity, degradation, and/or stoichiometry. Here we describe the preparation of novel polyleucine hydrophobic standards for SDS-PAGE that reduce the average deviation of the apparent MW from the formula MW of natural membrane proteins to 7% versus 20% with commercially available standards. Our results suggest that gel calibration with hydrophobic standards may facilitate the interpretation of membrane protein SDS-PAGE experiments.