Since the development of detection and analysis techniques for optical tweezers setups, there has been an ever-increasing interest in optical tweezers as a quantitative method, shifting its applications from a pure manipulation tool towards the investigation of motions and forces. With the capability of manipulation and detection of forces of a few hundred picoNewtons down to a fraction of a picoNewton, optical tweezers are perfectly suitable for the investigation of single molecules. Accordingly, the technique has been extensively used for the biophysical characterization of biomolecules, ranging from the mechanical and elastic properties of biological polymers to the dynamics associated with enzymatic activity and protein motility. Here, the use of state-of-the-art optical tweezers on the elasticity of single DNA molecules is presented, highlighting the possibilities this technique offers for the investigation of protein-DNA interaction, but also for other single molecule applications. Technical in nature, design aspects of the NanoTracker (TM) optical tweezers setup are addressed, presenting the recent advances in the development of optical tweezers, ranging from noise reduction to detection and calibration methodology.