The brain is the most complex and dynamically organized organ of the human body, with a high degree of computation capability enabling the execution of a wide spectrum of physiological processes and behaviors. In the past decades a large number of genomics studies have been undertaken to investigate brain function and brain disorders, but despite these efforts many of the underlying molecular mechanisms still remain largely unknown. The implementation of mass spectrometry based quantitative proteomics in recent years enabled to tap into condition-specific protein trafficking and protein interaction that are the key to organelle proteome (dys)function. The technology for neuroproteomics is still evolving; currently there are no standardized protocols. In this review we describe the most commonly used methods to prepare brain subcellular fractions suitable for proteomics analysis, and highlight the various approaches for quantitative neuroproteomics.