In order to reconstruct the ecosystem structure of chemosynthetic environments in the fossil record, geochemical proxies must be developed. Here, we present a suite of novel compound-specific isotope parameters for tracing chemosynthetic production with a focus on understanding nitrogen dynamics in deep-sea cold seep environments. We examined the chemosymbiotic bivalve Bathymodiolus childressi from three geographically distinct seep sites on the NE Atlantic Margin and compared isotope data to non-chemosynthetic littoral mussels to test whether water depth, seep activity, and/or mussel bed size are linked to differences in chemosynthetic production. The bulk isotope analysis of carbon (δ¹³C) and nitrogen (δ¹⁵N), and δ¹⁵N values of individual amino acids (δ¹⁵N_AA) in both gill and muscle tissues, as well as δ¹⁵N_AA-derived parameters including trophic level (TL), baseline δ¹⁵N value (δ¹⁵N_Phe), and a microbial resynthesis index (ΣV), were used to investigate specific geochemical signatures of chemosynthesis. Our results show that δ¹⁵N_AA values provide a number of new proxies for relative reliance on chemosynthesis, including TL, ∑V, and both δ¹⁵N values and molar percentages (Gly/Glu mol% index) of specific AA. Together, these parameters suggested that relative chemoautotrophy is linked to both degree of venting from seeps and mussel bed size. Finally, we tested a Bayesian mixing model using diagnostic AA δ¹⁵N values, showing that percent contribution of chemoautotrophic versus heterotrophic production to seep mussel nutrition can be directly estimated from δ¹⁵N_AA values. Our results demonstrate that δ¹⁵N_AA analysis can provide a new set of geochemical tools to better understand mixotrophic ecosystem function and energetics, and suggest extension to the study of ancient chemosynthetic environments in the fossil record.