Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.