ABSTRACT: Studies of lionfish feeding ecology seek to document the ecological impact of this invasive predatory species and determine which native prey species are at greatest risk. There are 2 common approaches to feeding ecology through gut content analysis: morphological identification to the lowest possible taxonomic rank and/or DNA barcoding of individual prey components in the stomach. The major disadvantage of both techniques is their inability to use advanced digested material. This study introduces next-generation sequencing to lionfish feeding ecology, employing DNA metabarcoding to analyze all components of the gut contents, including the previously unidentifiable portion. Sixty-three lionfish were caught from the inshore and offshore reefs of La Parguera, Puerto Rico. Stomach contents were separated into 2 sample components—a liquid (i.e. digested) and undigested tissue. A 313 bp region of the cytochrome oxidase subunit I (COI) gene was amplified from extracted DNA using specific primers for Caribbean reef fish. Samples were sequenced with an Illumina MiSeq platform, and the resulting 950+ sequences were compared against GenBank and the Barcode of Life Database to identify specimens at the lowest taxonomic level. Thirty-nine fish species from 16 families were identified (35 each in the digested and tissue fractions), including members of Pomacentridae, Acanthuridae, Gobiidae, Apogonidae, and Scaridae. Using the digested liquiform material proved efficient in detecting prey species, especially those that would have been missed with traditional methods.