ABSTRACT: Quantifying food web structure and interactions is important for understanding food web ecology, conservation biology and management. Trophic position (TP) is a useful metric for these purposes, describing an organism's hierarchical role within the food web. TP can be estimated using the ratio of nitrogen isotopes (𝛿15N) of specific amino acids within animal tissue. An important component of the TP calculation is the trophic discrimination factor (TDF), which represents the enrichment of 15N with each trophic step. Early work suggested a fixed TDF throughout the food chain, but subsequent studies indicate this may not be appropriate. This study focuses on a shelf marine food web in the Northwest Atlantic, where we used bulk and amino acid-specific 𝛿15N to determine TDFs for four feeding guilds spanning three trophic levels. Additionally, we compared TDFs between different tissues: muscle, liver, and homogenised whole-organism. TDFs calculated from amino acid 𝛿15N decreased with increasing TP: 6.9‰ (± 2.3) for zooplankton, 5.1‰ (± 1.5) for pelagic fish, 4.5‰ (± 1.0) for Atlantic cod and 3.1‰ (±1.0) for harp seals. This resulted from a combined effect of reduced 15N enrichment of trophic amino acids and 15N enrichment in the source amino acid phenylalanine among higher TP consumers. Finally, there was no evidence for differences in TDF between tissue types. By providing trophic guild-specific TDF values, our study enhances the accuracy of TP estimates and supports the development of amino acid isotope analysis as a tool for assessing food web structure across diverse taxa in the Northwest Atlantic.