The pituitary is the central endocrine gland that plays complex regulatory roles in growth, reproduction and metabolism of the body. The human pituitary tissue proteome has been the target of a number of investigations that applied various combinations of advanced separation techniques, mass spectrometry, and bioinformatics tools. This review describes the main features of the bioanalytical workflows used in pituitary proteomics, and summarizes major applications in pituitary proteome mapping, differential protein expression profiling in health and disease, and discovery of post-translational modifications in pituitary proteins.