We investigated the trophic structure and habitat use of ten cetacean species occurring in the oceanic waters of the western South Atlantic using naturally-occurring stable isotopes. We analysed δ¹⁵N in individual amino acids (AA) to estimate cetacean trophic position (TP) and to evaluate the spatial differences in baseline δ¹⁵N (source AAs). We adjusted cetacean bulk-skin δ¹³C and δ¹⁵Nfor the effect of trophic level using their estimated TPs, obtaining δ ¹³C_Adjusted and δ¹⁵N_Adjusted, respectively. These values were applied to estimate the overlap in the niche areas of cetacean baseline sources. Our analyses showed spatial segregation between Steno bredanensis and the remaining odontocetes, and the high δ¹⁵N in this species reflects its occurrence in neritic waters of the southern region. The highest TPs were observed in Physeter macrocephalus, Stenella attenuataand Globicephala melas, while the lowest TPs were reported for S. longirostris, S. clymene and Orcinus orca. Overall, source AA-δ¹⁵N showed similar patterns as those of baseline-δ¹⁵N (zooplankton) and were higher in species sampledin the southernmost region of the study area (e.g.,Delphinus delphis). Isotopic niche areas estimated using δ ¹³C_Adjusted and δ¹⁵N_Adjustedsuggested high overlap in foraging area between S. frontalis and Tursiops truncatus, with the latter occupying a higher TP. Our analyses of δ¹⁵N in AAs provide a unique insight into the trophic ecology, forage areas and spatial segregation in resource use amongthese cetacean populations. Additionally, our work provides AA-δ ¹⁵N baseline for future studies on the trophic ecology and habitat use of marine organisms in the western South Atlantic.