AbstractAquatic macroinvertebrates are central to lentic and lotic food webs, and characterizing the items they consume is often useful for studies of trophic ecology and food webs. Macroinvertebrate gut contents have been examined frequently by aquatic ecologists, but to our knowledge, no protocol describing how to conduct quantitative gut-content analysis is available. To fill this gap, we provide detailed methods for collecting, preserving, dissecting, identifying, and analyzing gut-content data. We also present a bootstrapping method to compute the number of particles needed to characterize a gut-content sample sufficiently. We anticipate that our description will help guide others in future studies of macroinvertebrate gut contents, trophic status, and foodweb ecology.