A major goal of cancer research is elucidating the molecular events underlying carcinogenesis, with the goal of discovering better diagnostic markers and therapeutic targets. Proteomics aims to facilitate this process by applying newly developed methods and advanced analytic tools, such as mass spectrometry, for the investigation of the protein complement en masse. Proteomics is the comprehensive study of proteins and is aimed at analyzing their structure, function, modifications, expression, interactions, and localization in complex biological systems. This article reviews the state-of-the art in mass spectrometry-based approaches and their application for cancer biomarker discovery and validation.