Combining the use of predators as biological samplers together with measurements of the stable isotopic ratios (d 13 C Bulk and d 15 N Bulk ) of their sclerotized beaks help investigate foraging ecology of poorly known oceanic cephalopods. However, high chitin content (an amino-sugar macromolecule) lowers beak d 15 N Bulk values, thus precluding direct isotopic comparison with other tissues and organisms. To overcome the chitin effect, compound-specific isotopic analysis of amino acids (CSIA-AA) was performed on squid beaks. The method was applied on beaks and muscle, and the resulting d 13 C AA and d 15 N AA values compared between tissues. The usefulness of CSIA was tested by defining the habitat and trophic position (TP CSIA ) of squids using their d 13 C AA and d 15 N AA values. Beak d 13 C AA values were reliably measured on 12 AA that included 5 essential and 7 non-essential AA, and d 15 N AA values were quantified on at least 7 AA that included 2 source and 4 trophic AA. Importantly, d 13 C AA and d 15 N AA varied little between muscle and lower and upper beaks, and TP CSIA estimates were identical regardless of the tissue considered. Tissue d 13 C AA values of both essential and non-essential AA reflected the latitudinal baseline d 13 C gradient that occurs in the Southern Indian Ocean, while beak d 15 N AA from source and trophic AA allowed the disentangling of the baseline effect from the trophic effect, and thus better calculations of squid TP estimates than from d 15 N Bulk values. Beak d 13 C AA and d 15 N AA defined isotopic niches of colossal and giant squids, the 2 largest living invertebrates. In subantarctic waters, they segregate by having species-specific foraging habitats (using d 13 C Gly or d 15 N Phe ) and TP CSIA (using d 15 N Glx and d 15 N Phe ). TP CSIA is higher in colossal (4.7) than giant (4.3) squids, and both values compare well with those of myctophid-eaters, suggesting very large squids prey primarily upon small zoo plankti vorous fishes. As expected, CSIA-AA overcomes the chitin effect on beaks and it is a powerful tool to investigate trophic interactions of cephalopods. The method has a great potential with arthropods, because chitin is a main component of their exoskeleton but the deleterious effect of chitin is overlooked in isotopic studies focusing on crustaceans and insects.