Membrane proteins play a central role in biological processes, but their separation and quantification using two‐dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin‐coated resin and proteolytically digested. The resulting peptides are analyzed by high‐pressure liquid chromatography and mass spectrometry, thus yielding a two‐dimensional peptide map. Matrix assisted laser desorption/ionization‐time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA‐spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial‐cadherin/actin/catenin complex, revealing an increased accumulation of beta‐catenin at 2% O2 concentration.