Here, we outline protocols to study cold acclimation in Drosophila from a neurobiological perspective, starting with fictive cold acclimation using a custom-built optogenetics-housing apparatus we call the OptoBox. We also provide detailed steps for single-unit electrophysiological recordings from larval cold nociceptors and a high-throughput cold-tolerance assay. These protocols expand the toolkit for the study of insect cold acclimation and nociception. For complete details on the use and execution of this protocol, please refer to Himmel et al. (2021).