The vast majority of proteomic studies to date have relied on mass spectrometric techniques to identify, and in some cases quantify, peptides that have been generated by proteolysis. Current approaches differ in the types of instrument used, their performance profiles, the manner in which they interface with biological research strategies, and their reliance on and use of prior information. Here, we consider the three main mass spectrometry (MS)-based proteomic approaches used today: shotgun (or discovery), directed and targeted strategies. We discuss the principles of each technique, their strengths and weaknesses and the dependence of their performance profiles on the composition of the biological sample. Our goal is to provide a rational framework for selecting strategies optimally suited to address the specific research issue under consideration.