Optical tweezers are flexible and powerful single-molecule tools that have been extensively utilized in biophysical studies. With their ability to stretch and twist DNA, and measure its force and torque simultaneously, they provide excellent opportunities to gain novel insights into the function of protein motors and protein-DNA interactions. Recently, a novel DNA supercoiling assay using an angular optical tweezers (AOT) has been developed to investigate torque generation during transcription. Here, we provide a detailed and practical guide to performing this technique. Using bacterial RNA polymerase (RNAP) as an example, we present protocols for constructing and calibrating an AOT instrument, preparing DNA templates, and acquiring and analyzing real-time data for transcription under DNA supercoiling. While these protocols were initially developed with E. coli RNAP, they can be readily adapted to study other DNA-based motor proteins. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.