Identifying a DNA extraction method that yields a high quantity and quality of DNA is a crucial component of molecular ecological studies; and the best suited method can vary greatly depending on research priorities. Here, we propose an extraction method which is suited for the non-destructive extraction of gut content DNA from single insect specimens with the goal of analyzing gut-associated microbiomes. We tested multiple factors, including the inclusion and exclusion of pre-lysis bleaching and post-lysis proteinase K inactivation. Two purification methods were used: a chelating beads-based method and a silica column-based method which served as a quality reference. Based on our findings, we recommend a method which includes pre-lysis bleaching, no proteinase K inactivation, and uses a chelating bead-based purification method. Our optimized protocol results in a high DNA yield suitable for downstream analyses including qPCR and next-generation sequencing.