Tissue microarray (TMA) technologies have been developed over the last years, mainly to identify biomarkers useful for the correct identification and characterization of tumors. Moreover, TMA has been implemented in retrospective studies in order to identify predictive biomarkers of response to a given therapy and/or to find potential new targets for biological therapy. We analyzed the fields of application of TMA technology and the design of TMA varying according to the objectives to be studied. In this article, the reader will learn how to design TMAs in order to cover the objectives of clinical trials based upon the use of target-based agents. The main limits and advantages of TMA and the results achieved in cancer diagnosis will be also described. Tissue microarray technology should be systematically applied to define critical markers, in retrospective studies and in the screening of most human tumors in order to find new possible molecular targets and to molecularly define the diagnosis of the neoplastic diseases. TMAs have substantially improved the field of translational studies, even in the design and follow-up of studies based upon the use of target-based agents in cancer therapy.