Abstract: Lipids in animal muscle tissue influence the determination results of stable isotopes. Given the high body fat content in cetaceans, this effect might be more pronounced, potentially leading to misinterpretations in their dietary source analysis. To investigate the impacts of lipid removal treatment on carbon and nitrogen stable isotope analysis in cetacean muscle tissue, we applied a methanol-chloroform lipid extraction method. We treated the muscle tissues from Indo-Pacific humpback dolphins (Sousa chinensis) and East Asian finless porpoises (Neophocaena asiaeorientalis sunameri) with lipid removal at six different time gradients, and fitted curves to track temporal shifts in carbon stable isotope values (δ¹³C). The results show that there was a significant difference in δ¹³C levels, but not in δ¹⁵N levels, in muscle tissue before and after lipid removal. And δ¹³C values in both species increased initially with longer extraction time but stabilized after approximately 6 h. Based on these findings, this study identified the optimal lipid removal duration for small cetaceans that balances accuracy and efficiency, providing a reference for subsequent related research. Finally, we treated the comparative analysis of carbon and nitrogen stable isotopes in muscle tissues of S. chinensis and N. asiaeorientalis sunameri for optimal lipid removal duration, as well as investigated niche differentiation between the two species, thereby further elucidating their trophic relationship. The results indicate a high degree of trophic niche overlap between the two species in the Taiwan Strait, with differentiation potentially occurring along other dimensions, such as habitat utilization.