Cancer proteomics is a rapidly developing field and promises to accelerate the discovery of new diagnostic, prognostic and therapy-related biomarkers. Increasingly, the mechanisms of action of cancer drugs are defined at the molecular level. The possibility of detecting hundreds and thousands of proteins at the same point of time is a tool to define proteins associated with response or resistance to therapy. This strategy has been applied successfully in cell culture models and is implemented as translational research tool in early clinical trials of new anti-cancer agents. This review aims to summarize basic concepts and techniques of proteome analysis, its challenges and limitations, and discusses the opportunities for cancer therapy.