Surrogate markers and biomarkers based on imaging readouts providing predictive information on clinical outcome are of increasing importance in the preclinical and clinical evaluation of novel therapies. They are primarily used in studies designed to establish evidence that the therapeutic principle is valid in a representative patient population or in an individual. A critical step in the development of (imaging) surrogates is validation: correlation with established clinical endpoints must be demonstrated. Biomarkers must not fulfill such stringent validation criteria; however, they should provide insight into mechanistic aspects of the therapeutic intervention (proof-of-mechanism) or document therapy efficacy with prognostic quality with regard to the long-term clinical outcome (proof of concept). Currently used imaging biomarkers provide structural, physiological and metabolic information. Novel imaging approaches annotate structure with molecular signatures that are tightly linked to the pathophysiology or to the therapeutic principle. These cellular and molecular imaging methods yield information on drug biodistribution, receptor expression and occupancy, and/or intra- and intercellular signaling. The design of novel target-specific imaging probes is closely related to the development of the therapeutic agents and should be considered early in the discovery phase. Significant technical and regulatory hurdles have to be overcome to foster the use of imaging biomarkers for clinical drug evaluation.