Per- and polyfluoroalkyl substances (PFAS) have gained significant global attention due to their extensive industrial use and harmful effects on various organisms. Among these, perfluoroalkyl acids (PFAAs) are well-studied, but their diverse precursors remain challenging to monitor. The Total Oxidizable Precursor (TOP) assay offers a powerful approach to converting these precursors into detectable PFAAs. In this study, the TOP assay was applied to samples from the East Asian-Australian Flyway, a critical migratory route for millions of shorebirds. Samples included shellfish from China's coastal mudflats, key stopover sites for these birds, and blood and liver samples from shorebirds overwintering in Australia. The results showed a substantial increase in perfluorocarboxylic acids (PFCAs) across all sample types following the TOP assay, with the most significant increases in shorebird livers (Sum PFCAs increased by 18,156 %). Intriguingly, the assay also revealed unexpected increases in perfluorosulfonic acids (PFSAs), suggesting the presence of unidentified precursors. These findings highlight the need for further research into these unknown precursors, their sources, and their ecological impacts on shorebirds, other wildlife, and potential human exposure. This study also provides crucial insights into the TOP assay's strengths and limitations in studying PFAS precursor dynamics in biological matrices.