Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. Dual-trap optical tweezers with differential detection have the advantage that the instrument is decoupled from sources of environmental noise, thereby minimizing interference of signal and maximizing stability and spatial resolution. In this article, we describe methods of data detection and calibration for our dual-trap design. Copyright © 2009 by Cold Spring Harbor Laboratory Press.