Many elasmobranch (shark and ray) species are considered threatened and their identification in processed products is important for conservation and authentication purposes. However, identification of elasmobranch species in shark cartilage pills has proven difficult using existing methodologies. The objective of this study was to develop a DNA mini-barcoding protocol using a ~130 bp region of the cytochrome c oxidase subunit I (COI) gene for species identification in shark cartilage pills. A total of 22 shark cartilage products underwent DNA extraction in duplicate using the DNeasy Blood and Tissue Kit (Qiagen). The effectiveness of a clean-up step following DNA extraction was analyzed by comparing DNA purity values and polymerase chain reaction (PCR) amplification rates. Next, five different mini-barcode primer sets were compared based on amplification rates, and the three top-performing primer sets were used in DNA sequencing. The incorporation of a clean-up step following DNA extraction showed a slight advantage over DNA extraction alone, with a higher amplification rate (52.3% vs. 47.8%) and A260/A230value (3.3 vs. 0.6). The three primer sets selected for DNA mini-barcoding showed DNA sequencing rates of 54.5–65.9% among the 44 duplicate samples. When the results for all three primer sets were combined, 18 of the 22 shark cartilage products were identified to the species or genus level. On an individual basis, the best-performing primer set identified 16 of the 22 products to the species or genus level. Overall, the protocol developed in this study increased the identification rate for elasmobranches in cartilage products by more than 2-fold as compared to previous research.