The major factors that have limited the use of DNA microarrays in research and diagnostics are the amount of target needed, the detection specificity, as well as the cost and reliability of detection equipment and assays. While the current gold standard for detection is fluorescence technology, the emphasis on more efficient and sensitive instrumentation has spurred the development of a number of new labeling and detection methodologies. Recent reports have demonstrated that alternative techniques like semiconductor or metal nanoparticle labels or electrochemical detection techniques could eliminate the need for target amplification steps such as PCR. Besides, the improvements in labeling technology enabled multiplexing by increasing the number of detectable colour channels and direct tracking of gene expression inside living cells.This chapter is devoted to the latest developments in terms of hybridization techniques and read-out technologies that are promising for enhanced detection and quantification of specific gene sequences. These techniques are widely used in two emerging trends: the bio-barcode strategy and live-cell imaging.