Optical trapping, or optical tweezers, is a physical technique that utilizes a highly focused laser beam to hold and manipulate microscopic or sub-microscopic objects, as well as measure forces produced, with nanometer displacement and piconewton force resolution. Optical trapping has been pivotal in unveiling the mechanistic underpinnings of biological systems, including but not limited to characterization of molecular motor proteins, DNA mechanics, and cell mechanosensing. This transformative technology and the subsequent groundbreaking biological discoveries led to its inventor, Arthur Ashkin, winning the 2018 Nobel Prize in Physics. While a large portion of optical trapping studies originated at the single molecule level, since the turn of the century, approaches have expanded to include constructing multi-component or ensemble protein studies, developing diagnostic tools, utilizing novel nanotechnologies such as DNA origami, coupling force sensing with other advanced instrumentation or imaging, and innovating analysis procedures. The purpose of this Methods Collection is to highlight advances in optical trapping biophysical assays, techniques, instrumentation, and data analysis methods to broaden access and lower the threshold for researchers interested in applying these techniques in their research.