Abstract We developed a method to characterize different classes of membrane proteins within a single experiment and using simple matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) analysis. After membrane solubilization with the nondenaturing detergent n ‐dodecyl‐β‐ D ‐maltoside, proteins were separated successively by gel filtration and ion‐exchange chromatography and finally by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). This procedure allowed to characterize 70 proteins from a membrane fraction enriched in plant vacuolar membrane ( Arabidopsis ), including integral proteins like the V0 complex of the H + ‐ATPase, the H + ‐pyrophosphatase or the glutathione S ‐conjugate ATPase AtMRP1, and peripheral proteins like the subunits of the catalytic V1 complex of the H + ‐ATPase. Approximately 60% of identified proteins were predicted to possess at least two trans‐membrane domains. Furthermore, proteins, with molecular masses ranging between 20 and 200 kDa were distributed into two populations with maximum frequencies at p I 5.3 and 8.9. Finally, this procedure appeared to allow the identification of proteins known to be minor in whole‐cell extracts like signaling or vesicular trafficking proteins. Almost 50% of the proteins identified were functionally unknown whereas the others confirmed that the plant vacuole is a multipurpose compartment.