Tissue microarrays (TMA) technology has been widely developed in the last years to perform high-throughput immunohistochemical analyses for all tissue-based oncology research. TMA has completely revolutionized biomedical research, mainly to identify prognostic and predictive biomarkers. Many cores of tumour from paraffin-fixed tissue are incorporated into a single paraffin block to obtain a single slide used to perform simultaneous analysis of molecular targets. Recently the use of TMA has been implemented in clinical settings, in order to provide cancer specific molecular data, needed to define the correct therapeutic strategy. The aim of this review was to analyse the fields of application of TMA technology in oncology, emphasizing the adequacy/inadequacy for the immunohistochemical detection of cancer stem cells (CSCs) in human tumours and to test the feasibility of TMA for investigation of biomarkers within the tumour microenvironments.