The human pineal gland regulates the day-night dynamics of multiple physiological processes, especially through the secretion of melatonin. Recently, using mass spectrometry-based proteomics and dedicated analysis tools, we have identified regulated proteins and signaling pathways that differ between day and night and/or between control and autistic pineal glands. This large-scale proteomic approach is the method of choice to study proteins in a biological system globally. This chapter proposes a protocol for large-scale analysis of the pineal gland proteome.