Neuroproteomic technologies are crucial for a deeper understanding of molecular mechanisms underlying neuronal plasticity and pathologies of the central nervous system. For a comprehensive neuroproteomic analysis, high-resolution high-throughput techniques are indispensable. The most commonly used-system for 2D gel electrophoresis is based on the combination of isoelectric focussing and sodium dodecyl-sulphate-polyacrylamide gel electrophoresis. Nevertheless, the analysis of complex samples derived from biochemically prepared synaptic fractions raises some crucial challenges. Therefore, we describe the-general 2D gel electrophoresis procedure, including sample preparation, gel casting, electrophoresis-condition, and the detection of proteins on the gel with mass spectrometry compatible silver staining, sypro-ruby or colloidal Coomassie Brilliant Blue.